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.

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 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.

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

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.

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.

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.

scholarly journals The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome

2005 ◽  
Vol 26 (2) ◽  
pp. 251-282 ◽  
Author(s):  
Héctor F. Escobar-Morreale ◽  
Manuel Luque-Ramírez ◽  
José L. San Millán
Keyword(s):  
Environmental Factors ◽  
Polycystic Ovary Syndrome ◽  
Diagnostic Criteria ◽  
Genetic Basis ◽  
Polycystic Ovary ◽  
Molecular Genetic ◽  
Ovary Syndrome ◽  
The Metabolic Syndrome ◽  
Molecular Genetic Basis ◽  
Functional Hyperandrogenism

The genetic mechanisms underlying functional hyperandrogenism and the polycystic ovary syndrome (PCOS) remain largely unknown. Given the large number of genetic variants found in association with these disorders, the emerging picture is that of a complex multigenic trait in which environmental influences play an important role in the expression of the hyperandrogenic phenotype. Among others, genomic variants in genes related to the regulation of androgen biosynthesis and function, insulin resistance, and the metabolic syndrome, and proinflammatory genotypes may be involved in the genetic predisposition to functional hyperandrogenism and PCOS. The elucidation of the molecular genetic basis of these disorders has been burdened by the heterogeneity in the diagnostic criteria used to define PCOS, the limited sample size of the studies conducted to date, and the lack of precision in the identification of ethnic and environmental factors that trigger the development of hyperandrogenic disorders. Progress in this area requires adequately sized multicenter collaborative studies after standardization of the diagnostic criteria used to classify hyperandrogenic patients, in whom modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision. In addition to classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, promising genomic and proteomic approaches will be paramount to our understanding of the pathogenesis of functional hyperandrogenism and PCOS, allowing a more precise prevention, diagnosis, and treatment of these prevalent disorders.

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