FUNCTIONAL STUDY OF TWO NEW POINT MUTATIONS OF EPOR GENE IN PRIMARY FAMILIAL CONGENITAL POLYCYTHEMIA
(Abstract release date: 05/19/16)
EHA Library. Randi M. 06/09/16; 134914; PB2014
Prof. Dr. Maria Luigia Randi
Contributions
Contributions
Abstract
Abstract: PB2014
Type: Publication Only
Background
Primary Familial Congenital Polycythemia (PFCP) is extremely rare. It is caused by mutations in the EPO receptor (EPOR) gene (locus 19p13.3-p13.2) resulting in hypersensitivity to erythropoietin EPO stimulus. In PFCP, in spite of reduced levels of serum EPO, mutated EPOR is ''switched on'' to stimulate red blood cell production by erythroid progenitor cells but without ''switch off'' mechanism.
Aims
Evaluate the functional effect of two EPOR gene novel mutations.
Methods
In our huge cohort of patients with erythrocytosis, we found 2 unrelated patients carrying novel mutations in EPOR gene. We performed a functional assay to evaluate the activation of EPOR signaling in these mutations: first we created mutated plasmids to transfect K562 cell as model. 48h after electroporation, we performed a stimulating assay with EPO following a time course at 0’, 5’ 15’ 30’ and 45’. We finally studied the signaling cascade activity with Western Blot analysis.
Results
Both observed mutations were found in exon 8 of EPOR gene: c.1013G>A, p.Cys338Tyr and c.1022C>T, p.Thr341Met.The EPOR signaling cascade resulted more active in mutated cells than in WT cells when stimulated with EPO. 5’ and 15’ after stimulation, transfected cells with C338Y mutation showed higher phosphorylation of STAT5 compared to T341M mutation.We observed also a similar phosphorylation pattern of ERK occurring earlier than STAT5 (at 0’ and 5’), while no signal of activity was detected in JAK2 and AKT cascades.
Conclusion
In this study, we report two new missense mutations of the EPOR gene, located in exon 8 as all the 22 previous described functional mutations. Exon 8 encodes the C-terminal negative regulatory domain of the protein. Both mutations here described impair the C-terminal negative regulatory domain of EPOR resulting in a gain-of-function in the EPOR signalling cascades, more relevant for C338Y than T341M mutation.These novel mutations, causing hyperactivity, increase proliferation and differentiation, and decrease of apoptosis of erythroid progenitor, sustain the erythrocytotic phenotype of our patients.
Session topic: E-poster
Keyword(s): Erythrocytosis, Erythropoietin receptor
Type: Publication Only
Background
Primary Familial Congenital Polycythemia (PFCP) is extremely rare. It is caused by mutations in the EPO receptor (EPOR) gene (locus 19p13.3-p13.2) resulting in hypersensitivity to erythropoietin EPO stimulus. In PFCP, in spite of reduced levels of serum EPO, mutated EPOR is ''switched on'' to stimulate red blood cell production by erythroid progenitor cells but without ''switch off'' mechanism.
Aims
Evaluate the functional effect of two EPOR gene novel mutations.
Methods
In our huge cohort of patients with erythrocytosis, we found 2 unrelated patients carrying novel mutations in EPOR gene. We performed a functional assay to evaluate the activation of EPOR signaling in these mutations: first we created mutated plasmids to transfect K562 cell as model. 48h after electroporation, we performed a stimulating assay with EPO following a time course at 0’, 5’ 15’ 30’ and 45’. We finally studied the signaling cascade activity with Western Blot analysis.
Results
Both observed mutations were found in exon 8 of EPOR gene: c.1013G>A, p.Cys338Tyr and c.1022C>T, p.Thr341Met.The EPOR signaling cascade resulted more active in mutated cells than in WT cells when stimulated with EPO. 5’ and 15’ after stimulation, transfected cells with C338Y mutation showed higher phosphorylation of STAT5 compared to T341M mutation.We observed also a similar phosphorylation pattern of ERK occurring earlier than STAT5 (at 0’ and 5’), while no signal of activity was detected in JAK2 and AKT cascades.
Conclusion
In this study, we report two new missense mutations of the EPOR gene, located in exon 8 as all the 22 previous described functional mutations. Exon 8 encodes the C-terminal negative regulatory domain of the protein. Both mutations here described impair the C-terminal negative regulatory domain of EPOR resulting in a gain-of-function in the EPOR signalling cascades, more relevant for C338Y than T341M mutation.These novel mutations, causing hyperactivity, increase proliferation and differentiation, and decrease of apoptosis of erythroid progenitor, sustain the erythrocytotic phenotype of our patients.
Session topic: E-poster
Keyword(s): Erythrocytosis, Erythropoietin receptor
Abstract: PB2014
Type: Publication Only
Background
Primary Familial Congenital Polycythemia (PFCP) is extremely rare. It is caused by mutations in the EPO receptor (EPOR) gene (locus 19p13.3-p13.2) resulting in hypersensitivity to erythropoietin EPO stimulus. In PFCP, in spite of reduced levels of serum EPO, mutated EPOR is ''switched on'' to stimulate red blood cell production by erythroid progenitor cells but without ''switch off'' mechanism.
Aims
Evaluate the functional effect of two EPOR gene novel mutations.
Methods
In our huge cohort of patients with erythrocytosis, we found 2 unrelated patients carrying novel mutations in EPOR gene. We performed a functional assay to evaluate the activation of EPOR signaling in these mutations: first we created mutated plasmids to transfect K562 cell as model. 48h after electroporation, we performed a stimulating assay with EPO following a time course at 0’, 5’ 15’ 30’ and 45’. We finally studied the signaling cascade activity with Western Blot analysis.
Results
Both observed mutations were found in exon 8 of EPOR gene: c.1013G>A, p.Cys338Tyr and c.1022C>T, p.Thr341Met.The EPOR signaling cascade resulted more active in mutated cells than in WT cells when stimulated with EPO. 5’ and 15’ after stimulation, transfected cells with C338Y mutation showed higher phosphorylation of STAT5 compared to T341M mutation.We observed also a similar phosphorylation pattern of ERK occurring earlier than STAT5 (at 0’ and 5’), while no signal of activity was detected in JAK2 and AKT cascades.
Conclusion
In this study, we report two new missense mutations of the EPOR gene, located in exon 8 as all the 22 previous described functional mutations. Exon 8 encodes the C-terminal negative regulatory domain of the protein. Both mutations here described impair the C-terminal negative regulatory domain of EPOR resulting in a gain-of-function in the EPOR signalling cascades, more relevant for C338Y than T341M mutation.These novel mutations, causing hyperactivity, increase proliferation and differentiation, and decrease of apoptosis of erythroid progenitor, sustain the erythrocytotic phenotype of our patients.
Session topic: E-poster
Keyword(s): Erythrocytosis, Erythropoietin receptor
Type: Publication Only
Background
Primary Familial Congenital Polycythemia (PFCP) is extremely rare. It is caused by mutations in the EPO receptor (EPOR) gene (locus 19p13.3-p13.2) resulting in hypersensitivity to erythropoietin EPO stimulus. In PFCP, in spite of reduced levels of serum EPO, mutated EPOR is ''switched on'' to stimulate red blood cell production by erythroid progenitor cells but without ''switch off'' mechanism.
Aims
Evaluate the functional effect of two EPOR gene novel mutations.
Methods
In our huge cohort of patients with erythrocytosis, we found 2 unrelated patients carrying novel mutations in EPOR gene. We performed a functional assay to evaluate the activation of EPOR signaling in these mutations: first we created mutated plasmids to transfect K562 cell as model. 48h after electroporation, we performed a stimulating assay with EPO following a time course at 0’, 5’ 15’ 30’ and 45’. We finally studied the signaling cascade activity with Western Blot analysis.
Results
Both observed mutations were found in exon 8 of EPOR gene: c.1013G>A, p.Cys338Tyr and c.1022C>T, p.Thr341Met.The EPOR signaling cascade resulted more active in mutated cells than in WT cells when stimulated with EPO. 5’ and 15’ after stimulation, transfected cells with C338Y mutation showed higher phosphorylation of STAT5 compared to T341M mutation.We observed also a similar phosphorylation pattern of ERK occurring earlier than STAT5 (at 0’ and 5’), while no signal of activity was detected in JAK2 and AKT cascades.
Conclusion
In this study, we report two new missense mutations of the EPOR gene, located in exon 8 as all the 22 previous described functional mutations. Exon 8 encodes the C-terminal negative regulatory domain of the protein. Both mutations here described impair the C-terminal negative regulatory domain of EPOR resulting in a gain-of-function in the EPOR signalling cascades, more relevant for C338Y than T341M mutation.These novel mutations, causing hyperactivity, increase proliferation and differentiation, and decrease of apoptosis of erythroid progenitor, sustain the erythrocytotic phenotype of our patients.
Session topic: E-poster
Keyword(s): Erythrocytosis, Erythropoietin receptor
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