Molecular Genetic Analysis of a Compound Heterozygote for the Glycoprotein (GP) IIb Gene Associated With Glanzmann’s Thrombasthenia: Disruption of the 674-687 Disulfide Bridge in GPIIb Prevents Surface Exposure of GPIIb-IIIa Complexes

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 866-875 ◽  
Author(s):  
Consuelo González-Manchón ◽  
Marta Fernández-Pinel ◽  
Elena G. Arias-Salgado ◽  
Milagros Ferrer ◽  
M.-Victoria Alvarez ◽  
...  
Keyword(s):  
Molecular Genetic ◽  
Splice Junction ◽  
Disulfide Bridge ◽  
Molecular Genetic Analysis ◽  
Pcr Analysis ◽  
Polymorphism Analysis ◽  
Glanzmann’S Thrombasthenia ◽  
Single Stranded Conformational Polymorphism ◽  
Glanzmann's Thrombasthenia ◽  
Platelet Content

Abstract This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann’s thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)–single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient’s GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C→A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C→A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband’s GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113→C transition that changes Cys674→Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.

Molecular Genetic Analysis of a Compound Heterozygote for the Glycoprotein (GP) IIb Gene Associated With Glanzmann’s Thrombasthenia: Disruption of the 674-687 Disulfide Bridge in GPIIb Prevents Surface Exposure of GPIIb-IIIa Complexes

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 866-875 ◽  
Author(s):  
Consuelo González-Manchón ◽  
Marta Fernández-Pinel ◽  
Elena G. Arias-Salgado ◽  
Milagros Ferrer ◽  
M.-Victoria Alvarez ◽  
...  
Keyword(s):  
Molecular Genetic ◽  
Splice Junction ◽  
Disulfide Bridge ◽  
Molecular Genetic Analysis ◽  
Pcr Analysis ◽  
Polymorphism Analysis ◽  
Glanzmann’S Thrombasthenia ◽  
Single Stranded Conformational Polymorphism ◽  
Glanzmann's Thrombasthenia ◽  
Platelet Content

This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann’s thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)–single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient’s GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C→A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C→A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband’s GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113→C transition that changes Cys674→Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.

scholarly journals The molecular genetic basis of Glanzmann's thrombasthenia in a gypsy population in France: identification of a new mutation on the alpha IIb gene

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 977-982 ◽  
Author(s):  
N Schlegel ◽  
O Gayet ◽  
MC Morel-Kopp ◽  
B Wyler ◽  
MF Hurtaud-Roux ◽  
...  
Keyword(s):  
Genetic Basis ◽  
Donor Site ◽  
Molecular Genetic ◽  
Premature Stop Codon ◽  
Splice Donor Site ◽  
Pcr Analysis ◽  
New Mutation ◽  
Glanzmann’S Thrombasthenia ◽  
Molecular Genetic Basis ◽  
Glanzmann's Thrombasthenia

Glanzmann's thrombasthenia is a rare inherited bleeding disorder caused by a qualitative or quantitative defect of platelet alpha IIb beta 3. We describe here a new mutation that is the molecular genetic basis of Glanzmann's thrombasthenia in two gypsy families. Our investigation was focused on the alpha IIb gene as a result of biochemical and immunologic analysis of patients' platelets showing undetectable alpha IIb but residual beta 3 levels. The entire alpha IIb cDNA was polymerase chain reaction (PCR) amplified using patients platelet RNA. Sequence analysis showed an 8-bp deletion located at the 3′ end of exon 15. This deletion causes a reading-frame shift leading to a premature stop codon and the synthesis of a severely truncated form of alpha IIb. Genomic DNA study showed a G-->A substitution, the Gypsy mutation, at the splice donor site of intron 15. This mutation results in an abnormal splicing occurring at an alternative donor site located 8 bp upstream from the mutation. Based on those results, an allele-specific PCR analysis was developed to allow a rapid identification of the mutation in patients and potential carriers of the gypsy community. This PCR analysis can also be used for genetic counseling and antenatal diagnosis.

scholarly journals The molecular genetic basis of Glanzmann's thrombasthenia in a gypsy population in France: identification of a new mutation on the alpha IIb gene

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 977-982 ◽  
Author(s):  
N Schlegel ◽  
O Gayet ◽  
MC Morel-Kopp ◽  
B Wyler ◽  
MF Hurtaud-Roux ◽  
...  
Keyword(s):  
Genetic Basis ◽  
Donor Site ◽  
Molecular Genetic ◽  
Premature Stop Codon ◽  
Splice Donor Site ◽  
Pcr Analysis ◽  
New Mutation ◽  
Glanzmann’S Thrombasthenia ◽  
Molecular Genetic Basis ◽  
Glanzmann's Thrombasthenia

Abstract Glanzmann's thrombasthenia is a rare inherited bleeding disorder caused by a qualitative or quantitative defect of platelet alpha IIb beta 3. We describe here a new mutation that is the molecular genetic basis of Glanzmann's thrombasthenia in two gypsy families. Our investigation was focused on the alpha IIb gene as a result of biochemical and immunologic analysis of patients' platelets showing undetectable alpha IIb but residual beta 3 levels. The entire alpha IIb cDNA was polymerase chain reaction (PCR) amplified using patients platelet RNA. Sequence analysis showed an 8-bp deletion located at the 3′ end of exon 15. This deletion causes a reading-frame shift leading to a premature stop codon and the synthesis of a severely truncated form of alpha IIb. Genomic DNA study showed a G-->A substitution, the Gypsy mutation, at the splice donor site of intron 15. This mutation results in an abnormal splicing occurring at an alternative donor site located 8 bp upstream from the mutation. Based on those results, an allele-specific PCR analysis was developed to allow a rapid identification of the mutation in patients and potential carriers of the gypsy community. This PCR analysis can also be used for genetic counseling and antenatal diagnosis.

Nonsense Mutation in Exon-19 of GPIIb Associated with Thrombasthenic Phenotype. Failure of GPIIb(Δ597-1008) to Form Stable Complexes with GPIIIa

2002 ◽  
Vol 87 (04) ◽  
pp. 684-691 ◽  
Author(s):  
Elena Arias-Salgado ◽  
Jianming Tao ◽  
Consuelo González-Manchón ◽  
Nora Butta ◽  
Vicente Vicente ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Secretory Pathway ◽  
Stop Codon ◽  
Molecular Genetic ◽  
Surface Expression ◽  
Molecular Genetic Analysis ◽  
Polymorphism Analysis ◽  
Type I ◽  
Single Stranded Conformational Polymorphism ◽  
Exon 19

SummaryWe report the molecular genetic analysis of a patient with thrombasthenic phenotype. The lack of surface platelet GPIIb-IIIa complexes and the presence of GPIIIa suggested it was a case of type I Glanzmann’s thrombasthenia due to a mutation in GPIIb. Single stranded conformational polymorphism analysis (SSCP) of exon-19 of GPIIb showed polymorphic DNA bands. The DNA sequence of exon-19 revealed the presence of a homozygous C1882T transition that changes residue R597 to STOP codon. Since no other mutations were found in either GPIIb or GPIIIa it is concluded that the C1882T substitution in GPIIb is responsible for the thrombasthenic phenotype of the patient. The lack of platelet GPIIb-mRNA in the proband indicates instability of the [C1882T]GPIIb-mRNA. Coexpression of normal GPIIIa and GPIIb(Δ597-1008) in CHO cells failed to show surface expression of GPIIb(Δ597-1008)-IIIa complexes. Immunoprecipitation analysis demonstrated that GPIIb(Δ597-1008) may indeed complex GPIIIa; however, the association is either unstable or incapable of progressing along the secretory pathway.

Abnormal Processing of the Glycoprotein IIb Transcript due to a Nonsense Mutation in Exon 17 Associated with Glanzmann’s Thrombasthenia

1995 ◽  
Vol 73 (05) ◽  
pp. 756-762 ◽  
Author(s):  
Yoshiaki Tomiyama ◽  
Hirokazu Kashiwagi ◽  
Satoru Kosugi ◽  
Masamichi Shiraga ◽  
Yoshio Kanayama ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Molecular Genetic ◽  
Genetic Defect ◽  
Nucleotide Sequence Analysis ◽  
Type I ◽  
Normal Amount ◽  
Immunoblot Assay ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Pcr Products

SummaryWe analyzed the molecular genetic defect responsible for type I Glanzmann’s thrombasthenia in a Japanese patient. In an immunoblot assay using polyclonal anti-GPIIb-IIIa antibodies, some GPIIIa (15% of normal amount) could be detected in the patient’s platelets, whereas GPIIb could not (<2% of normal amount). Nucleotide sequence analysis of platelet GPIIb mRNA-derived polymerase chain reaction (PCR) products revealed that patient’s GPIIb cDNA had a 75-bp deletion in the 3’ boundary of exon 17 resulting in an in-frame deletion of 25 amino acids. DNA analysis and family study revealed that the patient was a compound heterozygote of two GPIIb gene defects. One allele derived from her father was not expressed in platelets, and the other allele derived from her mother had a 9644C → T mutation which was located at the position -3 of the splice donor junction of exon 17 and resulted in a termination codon (TGA). Moreover, quantitative analysis demonstrated that the amount of the abnormal GPIIb transcript in the patient’s platelets was markedly reduced. Thus, the C → T mutation resulting in the abnormal splicing of GPIIb transcript and the reduction in its amount is responsible for Glanzmann’s thrombasthenia.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4178-4187 ◽  
Author(s):  
O. Peyruchaud ◽  
A.T. Nurden ◽  
S. Milet ◽  
L. Macchi ◽  
A. Pannochia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

R to Q Amino Acid Substitution in the GFFKR Sequence of the Cytoplasmic Domain of the Integrin IIb Subunit in a Patient With a Glanzmann’s Thrombasthenia-Like Syndrome

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4178-4187 ◽  
Author(s):  
O. Peyruchaud ◽  
A.T. Nurden ◽  
S. Milet ◽  
L. Macchi ◽  
A. Pannochia ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Chinese Hamster ◽  
Cytoplasmic Domain ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Polymorphism Analysis ◽  
Wild Type ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

The integrin IIbβ3 mediates platelet aggregation through its fibrinogen and adhesive protein-binding properties. Particular interest concerns the role of the cytoplasmic domains of IIb and β3. We now report the molecular analysis of IIbβ3 from a patient with a Glanzmann’s thrombasthenia-like syndrome for whom the principal characteristics are an approximate 50% total platelet content of IIbβ3 but with a much lower proportion in the surface pool (Hardisty et al, Blood 80:696, 1992). Polymerase chain reaction (PCR) single-strand conformational polymorphism and DNA sequencing showed a heterozygous mutation giving rise to amino acid substitution R995 to Q in the GFFKR sequence of the cytoplasmic domain of IIb. Reverse transcriptase-PCR and polymorphism analysis only detected mRNA for the mutated allele of the IIb gene and a single allele of the β3 gene in his platelets, suggesting other unidentified defects. Site-directed mutagenesis followed by transient expression of the mutated IIb together with wild-type β3 in Cos-7 cells resulted in a markedly decreased expression of the complex at the cell surface when compared with cells transfected with wild-type IIb and β3. Flow cytometry with PAC-1 and a stable Chinese hamster ovary–transfected cell line showed that the mutated receptor was not locked into a high activation state, although it became so in the presence of the activating antibody, anti-LIBS6. This is the first reported natural mutation in the highly conserved GFFKR sequence of the IIb cytoplasmic domain.

MOLECULAR TYPING OF METHICILLIN-SUSCEPTIBLE STAPHYLOCOCCUS AUREUS (MSSA), ISOLATED FROM MONKEYS, BASED ON COAGULASE GENE POLYMORPHISM

2019 ◽  
pp. 57-62
Author(s):  
V. A. Kalashnikova
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Molecular Genetic ◽  
Bovine Mastitis ◽  
Variable Region ◽  
Staphylococcal Infection ◽  
Pcr Analysis ◽  
Polymorphism Analysis ◽  
Coa Gene

Staphylococcus aureus is a very dangerous microorganism that causes more than 100 nosological forms of disease in humans and animals, including pneumonia, skin and soft tissue infections, food toxicoinfections, wound abscess, etc. Numerous studies on genotyping Staphylococcus aureus, isolated from humans, food and bovine mastitis have been carried out. The lack of information on the genotyping of these pathogens detected in monkeys living in captivity served as a stimulus to conduct a similar research, since staphylococcal infections in the primates are widespread. The present study is devoted to the study of the polymorphism of a variable region of the coagulase gene and to the typing of Staphylococcus aureus isolates from monkeys of different species kept at Adler monkey farm. 115 Staphylococcus aureus isolates were studied using phenotypic and molecular genetic methods. Genotyping was performed using PCR, real-time PCR and PCR with subsequent restriction fragment length polymorphism analysis (PCR-RFLP). A real-time PCR analysis allowed to classify all Staphylococcus aureus as methicillin-susceptible staphylococci (MSSA). After amplification of a variable region of the coagulase gene, 4 types of amplicons of 600, 700, 800, and 900 bp were generated. This data demonstrates structural differences of this gene in the studied isolates. The coagulase gene of 900 bp prevailed. The use of the Cfo1 endonuclease allowed to identify 23 different restriction profiles of the coa gene, but only three of them predominated. Staphylococcus aureus bacteria with seven types of coagulase gene were found only in the lungs of monkeys that died of pneumonia. The results obtained suggest that these isolates have tropism for lung tissue. Among Staphylococcus aureus isolated from pneumonia cases, isolates with three types of the coa gene prevailed. Staphylococcus aureus of eleven types of coagulase gene can be attributed to the invasive isolates, since they were detected in the tissues of various organs. Staphylococcal infection in monkeys kept at the monkey farm is caused by genotypically heterogeneous population of Staphylococcus aureus.

scholarly journals Glanzmann's thrombasthenia caused by homozygosity for a splice defect that leads to deletion of the first coding exon of the glycoprotein IIIa mRNA

Blood ◽  
1993 ◽  
Vol 81 (8) ◽  
pp. 2044-2049 ◽  
Author(s):  
S Simsek ◽  
H Heyboer ◽  
LG de Bruijne-Admiraal ◽  
R Goldschmeding ◽  
HT Cuijpers ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Donor Site ◽  
Molecular Genetic ◽  
Platelet Membrane ◽  
Splice Donor Site ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Inherited Disease ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract Glanzmann's thrombasthenia (GT) is the result of the absence or of an altered and dysfunctional expression on the platelet membrane of the fibrinogen receptor (glycoprotein [GP] IIb/IIIa complex). Various molecular genetic mechanisms have been found to be responsible for this inherited disease. In a patient with a severe type of GT, we have found a splice variant in the GP IIIa gene that leads to premature chain termination. Immunoprecipitation experiments, using monoclonal antibodies specific for GP IIb/IIIa, showed that GP IIb/IIIa was not detectable on the platelet membrane. Amplification of reversely transcribed platelet GP IIIa mRNA by the polymerase chain reaction and subsequent sequence analysis showed a 86-bp deletion, which corresponds to exon i of the GP IIIa gene. This deletion results in a shift of the reading frame leading to eight altered amino acids followed by a premature termination codon. Analysis of the corresponding genomic DNA fragments showed three mutations in the exon i-intron i boundary region of the GP IIIa gene. One of these mutations is a G-->T transition that eliminates the GT splice donor site in the wild type. This base pair change creates a restriction site for the enzyme Mse I. Allele-specific restriction enzyme analysis (ASRA) with Mse I of amplified genomic DNA of the parents and the proposita showed that both parents (who are first cousins) are heterozygous, whereas the proposita is homozygous for the G-->T substitution.

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