Contributions
Abstract: P423
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Genetics /Epigenetics and Pharmacogenetics
Much evidence implicates the P2X7 receptor and its “pore” function in promoting proinflammatory effects in the CNS including our recent genetic study showing a variant, Arg307Gln with loss of P2X7 pore function (but normal expression) was associated with two-fold protection against the risk of developing multiple sclerosis (MS) (Gu et al, Human Molecular Genetics, 2015). The P2X7 receptor however has a second function in the CNS as a scavenger receptor which recognizes and regulates the phagocytosis of apoptotic cells and foreign particles (such as beads) by cells of the monocyte-microglia lineage. We performed exome sequencing in five individuals from a single family affected with MS. Only a three variant P2RX7-P2RX4 haplotype (P2RX7 T205M N361S - P2RX4 G135S) was found in all five affected members, as well as in two obligate carriers and in one out of seven healthy relatives. Linkage analysis resulted in a LOD score of 3.07 for the whole family confirming co-segregation of the P2RX7-P2RX4 haplotype with disease. We then studied the functional effects on P2X7 function of each of the three variants separately and together as a haplotype. P2X7-induced “pore” formation was assessed by measuring ATP-induced ethidium uptake into HEK293 cells transfected with P2X7 and/or P2X4 constructs either wildtype or containing mutations to resemble the haplotype. The P2X7 205M-361S P2X4 135S constructs together conferred >95% inhibition of P2X7 “pore” function. Most but not all of the functional loss was associated with 205M alone. Surface expression of the P2X7 receptor was measured in transfected HEK293 cells by the binding of FITC conjugated monoclonal antibody specific for the extracellular domain of P2X7. The P2X7 205M-361S P2X4 135S constructs expressed together conferred >95% impairment of surface P2X7 expression. Finally we measured the phagocytosis of non-opsonized 1.0 um YO fluorescent beads by the transfected HEK293 cells. Cells transfected with wildtype P2X7 showed brisk uptake of beads while cells expressing P2X7 205M-361S P2X4 135S constructs showed major impairment of bead uptake. Our data show that variants with near total loss of surface expression of P2X7 receptors not only inhibit P2X7 “pore” function but also block the phagocytic function of this receptor. Loss of phagocytic function is a significant risk factor for developing MS.
Disclosure: Funded by the ARC and NHMRC of Australia.
A.L.T declares consultancy for Novartis, Genzyme and Roche. Other co-authors declare no conflict of interest.
Abstract: P423
Type: Poster
Abstract Category: Pathology and pathogenesis of MS - Genetics /Epigenetics and Pharmacogenetics
Much evidence implicates the P2X7 receptor and its “pore” function in promoting proinflammatory effects in the CNS including our recent genetic study showing a variant, Arg307Gln with loss of P2X7 pore function (but normal expression) was associated with two-fold protection against the risk of developing multiple sclerosis (MS) (Gu et al, Human Molecular Genetics, 2015). The P2X7 receptor however has a second function in the CNS as a scavenger receptor which recognizes and regulates the phagocytosis of apoptotic cells and foreign particles (such as beads) by cells of the monocyte-microglia lineage. We performed exome sequencing in five individuals from a single family affected with MS. Only a three variant P2RX7-P2RX4 haplotype (P2RX7 T205M N361S - P2RX4 G135S) was found in all five affected members, as well as in two obligate carriers and in one out of seven healthy relatives. Linkage analysis resulted in a LOD score of 3.07 for the whole family confirming co-segregation of the P2RX7-P2RX4 haplotype with disease. We then studied the functional effects on P2X7 function of each of the three variants separately and together as a haplotype. P2X7-induced “pore” formation was assessed by measuring ATP-induced ethidium uptake into HEK293 cells transfected with P2X7 and/or P2X4 constructs either wildtype or containing mutations to resemble the haplotype. The P2X7 205M-361S P2X4 135S constructs together conferred >95% inhibition of P2X7 “pore” function. Most but not all of the functional loss was associated with 205M alone. Surface expression of the P2X7 receptor was measured in transfected HEK293 cells by the binding of FITC conjugated monoclonal antibody specific for the extracellular domain of P2X7. The P2X7 205M-361S P2X4 135S constructs expressed together conferred >95% impairment of surface P2X7 expression. Finally we measured the phagocytosis of non-opsonized 1.0 um YO fluorescent beads by the transfected HEK293 cells. Cells transfected with wildtype P2X7 showed brisk uptake of beads while cells expressing P2X7 205M-361S P2X4 135S constructs showed major impairment of bead uptake. Our data show that variants with near total loss of surface expression of P2X7 receptors not only inhibit P2X7 “pore” function but also block the phagocytic function of this receptor. Loss of phagocytic function is a significant risk factor for developing MS.
Disclosure: Funded by the ARC and NHMRC of Australia.
A.L.T declares consultancy for Novartis, Genzyme and Roche. Other co-authors declare no conflict of interest.