NOVEL RARE VARIANTS IN EXON 4 OF GP6 HAVE LARGE EFFECTS ON GPVI EXPRESSION AND PLATELET FUNCTION
(Abstract release date: 05/19/16)
EHA Library. Sivapalaratnam S. 06/12/16; 135319; S825
Dr. Suthesh Sivapalaratnam
Contributions
Contributions
Abstract
Abstract: S825
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:30 - 08:45
Location: Room H4
Background
One of the initial steps in primary haemostasis is the binding of collagen by the GPVI signalling receptor on platelets. We have an interest in genetic variation in GP6 with an effect on expression and function. Platelets from GPVI-deficient patients are not activated by collagen, but do not show any severe bleeding phenotype. Therefore the GPVI receptor is a promising target for chronic anti-thrombotic treatment. Common variants in the GP6 gene are known to influence GPVI expression, but they only account for 16% of the observed variation seen in large cohort studies.
Aims
We hypothesized that a large proportion of the variation would be explained by rare variants in GP6 with a greater effect on expression and function.
Methods
The study was performed in the Cambridge Platelet Function cohort of 1500 health consented volunteers. GPVI function and expression was determined by flow cytometry and levels confirmed by Western blotting and ELISA. We planned on Sanger sequencing GP6 and FCER1G in the extreme cases with combined function and expression defects of GPVI. Where possible pedigrees of index cases with rare and functionally relevant GP6 variants, were recruited. Site-directed mutagenesis was performed to introduce individual identified variants and final plasmids verified by Sanger sequencing to test the functionality of the variants. The effect of the variants on thrombus formation was determined by the van Kruchten methodology.
Results
We identified two outlier individuals with approximately 50% reduction in GPVI expression levels and reduced functional responses. Sequencing in one of the individuals, identified a rare G/A variant at position 584 in the GP6 coding sequence, causing a serine to asparagine substitution at residue 195 (S195N). This variant had a frequency of 0.011% in 72.590 Caucasian individuals of the ExAC database. Sequencing of the second individual and their 4 pedigree members revealed a pattern consistent with the inheritance of a novel rare C/A variant at position 580 in the GP6 coding sequence, encoding a proline to threonine substitution at residue 194 (P194T). This variant was unobserved in the ExAC database. Both the S195N and P194T variants occur in a key structural motif of the second immunoglobulin-like domain of the receptor. In cell lines the S195N variant resulted in no expression of GPVI, whereas P194T did have expression. Both variants did effect thrombus formation in flowing whole blood over collagen-coated surfaces.
Conclusion
We have used a combination of platelet GPVI expression and function phenotypes, combined with sequencing of outliers, to identify rare variants with large effect on the native configuration of the cellular receptor and thereby its function and expression.
Session topic: Platelet disorders 2
Keyword(s): Genetic polymorphism, GPVI, Platelet function
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:30 - 08:45
Location: Room H4
Background
One of the initial steps in primary haemostasis is the binding of collagen by the GPVI signalling receptor on platelets. We have an interest in genetic variation in GP6 with an effect on expression and function. Platelets from GPVI-deficient patients are not activated by collagen, but do not show any severe bleeding phenotype. Therefore the GPVI receptor is a promising target for chronic anti-thrombotic treatment. Common variants in the GP6 gene are known to influence GPVI expression, but they only account for 16% of the observed variation seen in large cohort studies.
Aims
We hypothesized that a large proportion of the variation would be explained by rare variants in GP6 with a greater effect on expression and function.
Methods
The study was performed in the Cambridge Platelet Function cohort of 1500 health consented volunteers. GPVI function and expression was determined by flow cytometry and levels confirmed by Western blotting and ELISA. We planned on Sanger sequencing GP6 and FCER1G in the extreme cases with combined function and expression defects of GPVI. Where possible pedigrees of index cases with rare and functionally relevant GP6 variants, were recruited. Site-directed mutagenesis was performed to introduce individual identified variants and final plasmids verified by Sanger sequencing to test the functionality of the variants. The effect of the variants on thrombus formation was determined by the van Kruchten methodology.
Results
We identified two outlier individuals with approximately 50% reduction in GPVI expression levels and reduced functional responses. Sequencing in one of the individuals, identified a rare G/A variant at position 584 in the GP6 coding sequence, causing a serine to asparagine substitution at residue 195 (S195N). This variant had a frequency of 0.011% in 72.590 Caucasian individuals of the ExAC database. Sequencing of the second individual and their 4 pedigree members revealed a pattern consistent with the inheritance of a novel rare C/A variant at position 580 in the GP6 coding sequence, encoding a proline to threonine substitution at residue 194 (P194T). This variant was unobserved in the ExAC database. Both the S195N and P194T variants occur in a key structural motif of the second immunoglobulin-like domain of the receptor. In cell lines the S195N variant resulted in no expression of GPVI, whereas P194T did have expression. Both variants did effect thrombus formation in flowing whole blood over collagen-coated surfaces.
Conclusion
We have used a combination of platelet GPVI expression and function phenotypes, combined with sequencing of outliers, to identify rare variants with large effect on the native configuration of the cellular receptor and thereby its function and expression.
Session topic: Platelet disorders 2
Keyword(s): Genetic polymorphism, GPVI, Platelet function
Abstract: S825
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:30 - 08:45
Location: Room H4
Background
One of the initial steps in primary haemostasis is the binding of collagen by the GPVI signalling receptor on platelets. We have an interest in genetic variation in GP6 with an effect on expression and function. Platelets from GPVI-deficient patients are not activated by collagen, but do not show any severe bleeding phenotype. Therefore the GPVI receptor is a promising target for chronic anti-thrombotic treatment. Common variants in the GP6 gene are known to influence GPVI expression, but they only account for 16% of the observed variation seen in large cohort studies.
Aims
We hypothesized that a large proportion of the variation would be explained by rare variants in GP6 with a greater effect on expression and function.
Methods
The study was performed in the Cambridge Platelet Function cohort of 1500 health consented volunteers. GPVI function and expression was determined by flow cytometry and levels confirmed by Western blotting and ELISA. We planned on Sanger sequencing GP6 and FCER1G in the extreme cases with combined function and expression defects of GPVI. Where possible pedigrees of index cases with rare and functionally relevant GP6 variants, were recruited. Site-directed mutagenesis was performed to introduce individual identified variants and final plasmids verified by Sanger sequencing to test the functionality of the variants. The effect of the variants on thrombus formation was determined by the van Kruchten methodology.
Results
We identified two outlier individuals with approximately 50% reduction in GPVI expression levels and reduced functional responses. Sequencing in one of the individuals, identified a rare G/A variant at position 584 in the GP6 coding sequence, causing a serine to asparagine substitution at residue 195 (S195N). This variant had a frequency of 0.011% in 72.590 Caucasian individuals of the ExAC database. Sequencing of the second individual and their 4 pedigree members revealed a pattern consistent with the inheritance of a novel rare C/A variant at position 580 in the GP6 coding sequence, encoding a proline to threonine substitution at residue 194 (P194T). This variant was unobserved in the ExAC database. Both the S195N and P194T variants occur in a key structural motif of the second immunoglobulin-like domain of the receptor. In cell lines the S195N variant resulted in no expression of GPVI, whereas P194T did have expression. Both variants did effect thrombus formation in flowing whole blood over collagen-coated surfaces.
Conclusion
We have used a combination of platelet GPVI expression and function phenotypes, combined with sequencing of outliers, to identify rare variants with large effect on the native configuration of the cellular receptor and thereby its function and expression.
Session topic: Platelet disorders 2
Keyword(s): Genetic polymorphism, GPVI, Platelet function
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:30 - 08:45
Location: Room H4
Background
One of the initial steps in primary haemostasis is the binding of collagen by the GPVI signalling receptor on platelets. We have an interest in genetic variation in GP6 with an effect on expression and function. Platelets from GPVI-deficient patients are not activated by collagen, but do not show any severe bleeding phenotype. Therefore the GPVI receptor is a promising target for chronic anti-thrombotic treatment. Common variants in the GP6 gene are known to influence GPVI expression, but they only account for 16% of the observed variation seen in large cohort studies.
Aims
We hypothesized that a large proportion of the variation would be explained by rare variants in GP6 with a greater effect on expression and function.
Methods
The study was performed in the Cambridge Platelet Function cohort of 1500 health consented volunteers. GPVI function and expression was determined by flow cytometry and levels confirmed by Western blotting and ELISA. We planned on Sanger sequencing GP6 and FCER1G in the extreme cases with combined function and expression defects of GPVI. Where possible pedigrees of index cases with rare and functionally relevant GP6 variants, were recruited. Site-directed mutagenesis was performed to introduce individual identified variants and final plasmids verified by Sanger sequencing to test the functionality of the variants. The effect of the variants on thrombus formation was determined by the van Kruchten methodology.
Results
We identified two outlier individuals with approximately 50% reduction in GPVI expression levels and reduced functional responses. Sequencing in one of the individuals, identified a rare G/A variant at position 584 in the GP6 coding sequence, causing a serine to asparagine substitution at residue 195 (S195N). This variant had a frequency of 0.011% in 72.590 Caucasian individuals of the ExAC database. Sequencing of the second individual and their 4 pedigree members revealed a pattern consistent with the inheritance of a novel rare C/A variant at position 580 in the GP6 coding sequence, encoding a proline to threonine substitution at residue 194 (P194T). This variant was unobserved in the ExAC database. Both the S195N and P194T variants occur in a key structural motif of the second immunoglobulin-like domain of the receptor. In cell lines the S195N variant resulted in no expression of GPVI, whereas P194T did have expression. Both variants did effect thrombus formation in flowing whole blood over collagen-coated surfaces.
Conclusion
We have used a combination of platelet GPVI expression and function phenotypes, combined with sequencing of outliers, to identify rare variants with large effect on the native configuration of the cellular receptor and thereby its function and expression.
Session topic: Platelet disorders 2
Keyword(s): Genetic polymorphism, GPVI, Platelet function
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