ABSTRACT
Introduction: Extracellular ATP (eATP) released from damaged cells activates the P2X7 receptor (P2X7R) ion channel on the surface of surrounding cells, resulting in calcium influx, potassium efflux and inflammasome activation. Inherited changes in the P2X7R gene (P2RX7) influence eATP induced responses. Single nucleotide polymorphisms (SNPs) of P2RX7 influence both function and signaling of the receptor, that in addition to ion flux includes pathogen control and immunity. Methods: Subjects (n = 105) were admitted to the ICU at the University Hospital Ulm, Germany between June 2018 and August 2019. Of these, subjects with a diagnosis of sepsis (n = 75), were also diagnosed with septic shock (n = 24), and/or pneumonia (n = 42). Subjects with pneumonia (n = 43) included those without sepsis (n = 1), sepsis without shock (n = 29) and pneumonia with septic shock (n = 13). Out of the 75 sepsis/septic shock patients, 33 patients were not diagnosed with pneumonia. Controls (n = 30) were recruited to the study from trauma patients and surgical patients without sepsis, septic shock, or pneumonia. SNP frequencies were determined for 16 P2RX7 SNPs known to affect P2X7R function, and association studies were performed between frequencies of these SNPs in sepsis, septic shock, and pneumonia compared to controls. Results: The loss-of-function (LOF) SNP rs17525809 (T253C) was found more frequently in patients with septic shock, and non-septic trauma patients when compared to sepsis. The LOF SNP rs2230911 (C1096G) was found to be more frequent in patients with sepsis and septic shock than in non-septic trauma patients. The frequencies of these SNPs were even higher in sepsis and septic patients with pneumonia. The current study also confirmed a previous study by our group that showed a five SNP combination that included the GOF SNPs rs208294 (C489T) and rs2230912 (Q460R) that was designated #21211 was associated with increased odds of survival in severe sepsis. Discussion: The results found an association between expression of LOF P2RX7 SNPs and presentation to the ICU with sepsis, and septic shock compared to control ICU patients. Furthermore, frequencies of LOF SNPs were found to be higher in sepsis patients with pneumonia compared to those without pneumonia. In addition, a five SNP GOF combination was associated with increased odds of survival in severe sepsis. These results suggest that P2RX7 is required to control infection in pneumonia and that inheritance of LOF variants increases the risk of sepsis when associated with pneumonia. This study confirms that P2RX7 genotyping in pneumonia may identify patients at risk of developing sepsis. The study also identifies P2X7R as a target in sepsis associated with an excessive immune response in subjects with GOF SNP combinations.
Subject(s)
Pneumonia , Polymorphism, Single Nucleotide , Receptors, Purinergic P2X7 , Sepsis , Shock, Septic , Humans , Receptors, Purinergic P2X7/genetics , Male , Female , Shock, Septic/genetics , Shock, Septic/mortality , Shock, Septic/immunology , Middle Aged , Pneumonia/genetics , Pneumonia/mortality , Aged , Sepsis/genetics , Sepsis/mortality , Genetic Predisposition to Disease , Adenosine Triphosphate/metabolism , Adult , Aged, 80 and overABSTRACT
B cells are central to the adaptive immune response and provide long-lasting immunity after infection. B cell activation is mediated by the surface membrane-bound B cell receptor (BCR) following recognition of a specific antigen. The BCR has been challenging to analyse using mass spectrometry (MS) due to the difficulty of isolating and enriching this membrane-bound protein complex. There are approximately 120,000 BCRs on the B cell surface; however, depending on the B cell activation state, there may be hundreds-of-millions to billions of proteins in a B cell. Consequently, advanced proteomic techniques such as MS workflows that use purified proteins to yield structural and protein-interaction information have not been published for the BCR complex. This paper describes a method for enriching the BCR complex that is MS-compatible. The method involves a Protein G pull down on agarose beads using an intermediary antibody to each of the BCR complex subcomponents (CD79a, CD79b, and membrane immunoglobulin). The enrichment process is shown to pull down the entire BCR complex and has the advantage of being readily compatible with further proteomic study including MS analysis. Using intermediary antibodies has the potential to enrich all isotypes of the BCR, unlike previous methods described in the literature that use protein G-coated beads to directly pull down the membrane IgG (mIgG) but cannot be used for other mIg isotypes.
ABSTRACT
P2X7 receptors (P2X7Rs) are membrane-bound ATP-gated ion channels that are composed of three subunits. Different subunit structures may be expressed due to alternative splicing of the P2RX7 gene, altering the receptor's function when combined with the wild-type P2X7A subunits. In this study, the application of the deep-learning method, AlphaFold2-Multimer (AF2M), for the generation of trimeric P2X7Rs was validated by comparing an AF2M-generated rat wild-type P2X7A receptor with a structure determined by cryogenic electron microscopy (cryo-EM) (Protein Data Bank Identification: 6U9V). The results suggested AF2M could firstly, accurately predict the structures of P2X7Rs and secondly, accurately identify the highest quality model through the ranking system. Subsequently, AF2M was used to generate models of heterotrimeric alternatively spliced P2X7Rs consisting of one or two wild-type P2X7A subunits in combination with one or two P2X7B, P2X7E, P2X7J, and P2X7L splice variant subunits. The top-ranking models were deemed valid based on AF2M's confidence measures, stability in molecular dynamics simulations, and consistent flexibility of the conserved regions between the models. The structure of the heterotrimeric receptors, which were missing key residues in the ATP binding sites and carboxyl terminal domains (CTDs) compared to the wild-type receptor, help to explain their observed functions. Overall, the models produced in this study (available as supplementary material) unlock the possibility of structure-based studies into the heterotrimeric P2X7Rs.
ABSTRACT
B cells are central to the adaptive immune response, providing long lasting immunity after infection. B cell activation is mediated by a cell surface B cell receptor (BCR) following recognition of an antigen. BCR signaling is modulated by several co-receptors including CD22 and a complex that contains CD19 and CD81. Aberrant signaling through the BCR and co-receptors promotes the pathogenesis of several B cell malignancies and autoimmune diseases. Treatment of these diseases has been revolutionized by the development of monoclonal antibodies that bind to B cell surface antigens, including the BCR and its co-receptors. However, malignant B cells can escape targeting by several mechanisms and until recently, rational design of antibodies has been limited by the lack of high-resolution structures of the BCR and its co-receptors. Herein we review recently determined cryo-electron microscopy (cryo-EM) and crystal structures of the BCR, CD22, CD19 and CD81 molecules. These structures provide further understanding of the mechanisms of current antibody therapies and provide scaffolds for development of engineered antibodies for treatment of B cell malignancies and autoimmune diseases.
ABSTRACT
The P2X7 receptor (P2X7R) is an ATP-gated membrane ion channel that is expressed by multiple cell types. Following activation by extracellular ATP, the P2X7R mediates a broad range of cellular responses including cytokine and chemokine release, cell survival and differentiation, the activation of transcription factors, and apoptosis. The P2X7R is made up of three P2X7 subunits that contain specific domains essential for the receptor's varied functions. Alternative splicing produces P2X7 isoforms that exclude one or more of these domains and assemble in combinations that alter P2X7R function. The modification of the structure and function of the P2X7R may adversely affect cellular responses to carcinogens and pathogens, and alternatively spliced (AS) P2X7 isoforms have been associated with several cancers. This review summarizes recent advances in understanding the structure and function of AS P2X7 isoforms and their associations with cancer and potential role in modulating the inflammatory response.
Subject(s)
Neoplasms , Receptors, Purinergic P2X7 , Adenosine Triphosphate/metabolism , Cytokines/metabolism , Humans , Neoplasms/genetics , Protein Isoforms/genetics , Protein Isoforms/metabolism , Receptors, Purinergic P2X7/geneticsABSTRACT
INTRODUCTION: People with a family history of chronic lymphocytic leukemia (F-CLL) have an increased risk of monoclonal B lymphocytosis (F-MBL), which is found in up to 18% of first-degree relatives of patients compared to 5% of the total population. This may indicate that the presence of an F-MBL in the relative of a F-CLL patient is due to genetic susceptibility. In this study, we hypothesized that progressive changes in gene expression result in malignant transformation of B lymphocytes to F-MBL, and subsequent alterations in gene expression occur before overt F-CLL develops. The aim of this study of affected and unaffected individuals from a family with multiple CLL cases was to compare mRNA expression levels in control B-lymphocytes, pre-malignant F-MBL and malignant F-CLL cells. METHODS: To identify inherited changes in gene expression, a high-resolution DNA microarray was used to identify differentially abundant mRNAs in age-matched cases of F-MBL (n = 4), F-CLL (n = 2) and unaffected family relatives (F-Controls, n = 3) within one family. These were then compared to non-kindred controls (NK-Controls, n = 3) and sporadic CLL (S-CLL) cases (n = 6). RESULTS: Seven differentially abundant mRNAs were identified against similar genetic backgrounds of the family: GRASP and AC016745.3 were decreased in F-MBL and further decreased in F-CLL compared to F-Controls, whereas C11orf80 and METTL8 were progressively increased. PARP3 was increased in F-MBL compared to F-Controls but was decreased in F-CLL compared to F-MBL. Compared to F-Controls, levels of ROR1 and LEF1 were similarly increased in F-MBL and F-CLL. For six of the genes, there were no differences in mRNA levels between S-CLL and F-CLL; however PARP3 was higher in S-CLL. CONCLUSION: These results are consistent with the hypothesis that changes in expression of specific genes contribute to transformation from normal lymphocytes to MBL and CLL.
ABSTRACT
P2X7 is an ATP-gated membrane ion channel that is expressed by multiple cell types. Brief exposure to ATP induces the opening of a nonselective cation channel; while repeated or prolonged exposure induces formation of a transmembrane pore. This process may be partially regulated by alternative splicing of full-length P2RX7A pre-mRNA, producing isoforms that delete or retain functional domains. Here, we report cloning and expression of a novel P2RX7 splice variant, P2RX7L, that is, characterized by skipping of exons 7 and 8. In HEK 293 cells, expression of P2RX7L produces a protein isoform, P2X7L, that forms a heteromer with P2X7A. A haplotype defined by six single nucleotide polymorphisms (SNPs) (rs208307, rs208306, rs36144485, rs208308, rs208309, and rs373655596) promotes allele-specific alternative splicing, increasing mRNA levels of P2RX7L and another isoform, P2RX7E, which in addition has a truncated C-terminus. Skipping of exons 7 and 8 is predicted to delete critical amino acids in the ATP-binding site. P2X7L-transfected HEK 293 cells have phagocytic but not channel, pore, or membrane-blebbing function, and double-transfected P2X7L and P2X7A cells have reduced pore function. Heteromeric receptor complexes of P2X7A and P2X7L are predicted to have reduced numbers of ATP-binding sites, which potentially alters receptor function compared to homomeric P2X7A complexes.
Subject(s)
Exons/genetics , Polymorphism, Single Nucleotide/genetics , Receptors, Purinergic P2X7/genetics , Adult , Aged , Binding Sites/genetics , Blotting, Western , Cells, Cultured , Electrophysiology , Female , HEK293 Cells , Haplotypes/genetics , Humans , Male , Middle Aged , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: Use of antidepressant medications has been linked to detrimental impacts on bone mineral density and osteoporosis; however, the cellular basis behind these observations remains poorly understood. The effect does not appear to be homogeneous across the whole class of drugs and may be linked to affinity for the serotonin transporter system. In this study, we hypothesized that antidepressants have a class- and dose-dependent effect on mesenchymal stem cell (MSC) differentiation, which may affect bone metabolism. METHODS: Human MSCs (hMSCs) were committed to differentiate when either adipogenic or osteogenic media was added, supplemented with five increasing concentrations of amitriptyline (0.001-10 µM), venlafaxine (0.01-25 µM), or fluoxetine (0.001-10 µM). Alizarin red staining (mineralization), alkaline phosphatase (osteoblastogenesis), and oil red O (adipogenesis) assays were performed at timed intervals. In addition, cell viability was assessed using a MTT. RESULTS: We found that fluoxetine had a significant inhibitory effect on mineralization. Furthermore, adipogenic differentiation of hMSC was affected by the addition of amitriptyline, venlafaxine, and fluoxetine to the media. Finally, none of the tested medications significantly affected cell survival. CONCLUSIONS: This study showed a divergent effect of three antidepressants on hMSC differentiation, which appears to be independent of class and dose. As fluoxetine and amitriptyline, but not venlafaxine, affected both osteoblastogenesis and adipogenesis, this inhibitory effect could be associated to the high affinity of fluoxetine to the serotonin transporter system.
ABSTRACT
Adamantanyl benzamide 1 was identified as a potent P2X7R antagonist but failed to progress further due to poor metabolic stability. We describe the synthesis and SAR of a series of bioisosteres of benzamide 1 to explore improvements in the pharmacological properties of this lead. Initial efforts investigated a series of heteroaromatic bioisosteres, which demonstrated improved physicochemical properties but reduced P2X7R antagonism. Installation of bioisosteric fluorine on the adamantane bridgeheads was well tolerated and led to a series of bioisosteres with improved physicochemical properties and metabolic stability. Trifluorinated benzamide 34 demonstrated optimal physicochemical parameters, superior metabolic stability (ten times longer than lead benzamide 1), and an improved physicokinetic profile and proved effective in the presence of several known P2X7R polymorphisms.
Subject(s)
Adamantane/analogs & derivatives , Benzamides/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X7/drug effects , Adamantane/pharmacology , Animals , Benzamides/chemical synthesis , Benzamides/chemistry , Benzamides/pharmacokinetics , Biotransformation , Drug Evaluation, Preclinical , Drug Stability , Humans , Microsomes, Liver/metabolism , Molecular Structure , Oxidation-Reduction , Polymorphism, Single Nucleotide , Purinergic P2X Receptor Antagonists/chemical synthesis , Purinergic P2X Receptor Antagonists/chemistry , Purinergic P2X Receptor Antagonists/pharmacokinetics , Rats , Receptors, Purinergic P2X7/genetics , Structure-Activity RelationshipABSTRACT
P2X7 receptor (P2X7) activity may link inflammation to depressive disorders. Genetic variants of human P2X7 have been linked with major depression and bipolar disorders, and the P2X7 knockout mouse has been shown to exhibit anti-depressive-like behaviour. P2X7 is an ATP-gated ion channel and is a major regulator of the pro-inflammatory cytokine interleukin 1ß (IL-1ß) secretion from monocytes and microglia. We hypothesised that antidepressants may elicit their mood enhancing effects in part via modulating P2X7 activity and reducing inflammatory responses. In this study, we determined whether common psychoactive drugs could affect recombinant and native human P2X7 responses in vitro. Common antidepressants demonstrated opposing effects on human P2X7-mediated responses; paroxetine inhibited while fluoxetine and clomipramine mildly potentiated ATP-induced dye uptake in HEK-293 cells stably expressing recombinant human P2X7. Paroxetine inhibited dye uptake mediated by human P2X7 in a concentration-dependent manner with an IC(50) of 24 µM and significantly reduces ATP-induced inward currents. We confirmed that trifluoperazine hydrochloride suppressed human P2X7 responses (IC(50) of 6.4 µM). Both paroxetine and trifluoperazine did not inhibit rodent P2X7 responses, and mutation of a known residue (F 95L) did not alter the effect of either drug, suggesting neither drug binds at this site. Finally, we demonstrate that P2X7-induced IL-1ß secretion from lipopolysaccharide (LPS)-primed human CD14(+) monocytes was suppressed with trifluoperazine and paroxetine.
Subject(s)
Antidepressive Agents, Second-Generation/pharmacology , Paroxetine/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X7/drug effects , Animals , Antidepressive Agents, Tricyclic/pharmacology , Antipsychotic Agents/pharmacology , Clomipramine/pharmacology , Dose-Response Relationship, Drug , Fluoxetine/pharmacology , Humans , Interleukin-1beta/metabolism , Lipopolysaccharide Receptors/metabolism , Mice , Monocytes/drug effects , Monocytes/metabolism , Purinergic P2X Receptor Agonists/pharmacology , Rats , Recombinant Proteins/drug effects , Trifluoperazine/pharmacologyABSTRACT
The relative function of the P2X7 receptor, an ATP-gated ion channel, varies between humans due to polymorphisms in the P2RX7 gene. This study aimed to assess the functional impact of P2X7 variation in a random sample of the canine population. Blood and genomic DNA were obtained from 69 dogs selected as representatives of a cross section of different breeds. P2X7 function was determined by flow cytometric measurements of dye uptake and patch-clamp measurements of inward currents. P2X7 expression was determined by immunoblotting and immunocytochemistry. Sequencing was used to identify P2RX7 gene polymorphisms. P2X7 was cloned from an English springer spaniel, and point mutations were introduced into this receptor by site-directed mutagenesis. The relative function of P2X7 on monocytes varied between individual dogs. The canine P2RX7 gene encoded four missense polymorphisms: F103L and P452S, found in heterozygous and homozygous dosage, and R270C and R365Q, found only in heterozygous dosage. Moreover, R270C and R365Q were associated with the cocker spaniel and Labrador retriever, respectively. F103L, R270C, and R365Q but not P452S corresponded to decreased P2X7 function in monocytes but did not explain the majority of differences in P2X7 function between dogs, indicating that other factors contribute to this variability. Heterologous expression of site-directed mutants of P2X7 in human embryonic kidney-293 cells indicated that the R270C mutant was nonfunctional, the F103L and R365Q mutants had partly reduced function, and the P452S mutant functioned normally. Taken together, these data highlight that a R270C polymorphism has major functional impact on canine P2X7.
Subject(s)
Mutation, Missense/genetics , Polymorphism, Single Nucleotide/genetics , Receptors, Purinergic P2X7/genetics , Animals , Cell Line , Dogs , HEK293 Cells , Heterozygote , Homozygote , Humans , Madin Darby Canine Kidney Cells , Monocytes/metabolismABSTRACT
P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1ß secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 µM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1ß secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.
Subject(s)
Connexins/metabolism , Nerve Tissue Proteins/metabolism , Probenecid/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X7/metabolism , Adenosine Triphosphate/physiology , Biological Transport, Active , Calcium Signaling , Ethidium/metabolism , Fluorescent Dyes/metabolism , HEK293 Cells , Humans , Inhibitory Concentration 50 , Interleukin-1beta/metabolism , Isoquinolines/metabolism , Lipopolysaccharides/physiology , Monocytes/immunology , Monocytes/metabolismABSTRACT
Phagocytosis is central to immunity however a rapid and standardized method is much needed for quantitative assessment of the phagocytic process. We describe a real-time flow cytometric method to quantitate the phagocytosis of fluorescent latex beads by human monocytes in serum-free conditions. Effects of buffer composition, temperature, pH, and bead surface on phagocytic rate are described. The innate phagocytic ability of human monocytes from single subjects measured by this method was relatively stable over many months although phagocytosis rate varied as much as two-fold between individuals. Comparable results were obtained with a simplified method using several mL of whole blood which is suitable for routine clinical application. This method also allows two-color flow cytometric measurement of cytosolic calcium levels during the phagocytic uptake of fluorescent beads.
Subject(s)
Flow Cytometry/methods , Monocytes/immunology , Phagocytosis/physiology , Humans , Monocytes/metabolismABSTRACT
We report an alternative approach to colony screening using real-time PCR (qPCR) which can be used instead of the traditional end-point PCR to eliminate false-positives and reduce processing times. False-positive transformants can easily be distinguished from true-positives by comparing Ct values derived from qPCR amplification curves. In addition, the use of qPCR allows for more efficient processing since a gel electrophoresis step is not required and the screening process is no longer limited by the capacity of the gel apparatus.
Subject(s)
Bacteriological Techniques/methods , Molecular Diagnostic Techniques/methods , Real-Time Polymerase Chain Reaction/methods , False Positive Reactions , Genetic Testing/methods , Genetics, Microbial/methods , Transformation, BacterialABSTRACT
INTRODUCTION: The aim of this study was to examine the association between functional polymorphisms in the pro-inflammatory P2X7 receptor and the Ro/La autoantibody response in primary Sjögren's syndrome (pSS). METHODS: Twelve functional P2RX7 polymorphisms were genotyped in 114 pSS patients fulfilling the Revised American-European Consensus Criteria for pSS, and 136 controls. Genotyping of the A1405G (rs2230912) polymorphism was performed on a replication cohort consisting of 281 pSS patients and 534 controls. P2X7 receptor function in lymphocytes and monocytes was assessed by measurement of ATP-induced ethidium+ uptake. Serum IL-18 levels were determined by ELISA. RESULTS: The minor allele of P2RX7 A1405G is a tag for a common haplotype associated with gain in receptor function, as assessed by ATP-induced ethidium+ uptake. A positive association between 1405G and anti-Ro±La seropositive pSS patients was observed in Cohort 1. Although not replicated in Cohort 2, there was a consistent, significant, negative epistatic interaction effect with HLA-DR3 in seropositive pSS patients from both cohorts, thereby implicating this gain of function variant in the pathogenesis of pSS. Serum IL-18 was elevated in seropositive pSS patients, but was not influenced by P2RX7 A1405G. CONCLUSIONS: The P2RX7 1405G gain-of-function haplotype may be a risk factor for seropositive pSS in a subset of subjects who do not carry HLA risk alleles, but has no effect in subjects who do (epistasis). Potential mechanisms relate to autoantigen exposure and inflammatory cytokine expression. The observed elevation of IL-18 levels is consistent with P2X7 receptor activation in seropositive pSS patients. Collectively these findings implicate P2X7 receptor function in the pathogenesis of pSS.
Subject(s)
Epistasis, Genetic/genetics , HLA-DR3 Antigen/genetics , Receptors, Purinergic P2X7/genetics , Sjogren's Syndrome/genetics , Antibodies, Antinuclear/blood , Autoantibodies/blood , Autoantigens/immunology , Enzyme-Linked Immunosorbent Assay , Female , Genotype , Humans , Interleukin-18/blood , Male , Polymorphism, Single Nucleotide , Ribonucleoproteins/immunology , Sjogren's Syndrome/blood , Sjogren's Syndrome/immunology , SS-B AntigenABSTRACT
Age-related macular degeneration (AMD) is a leading cause of blindness in Western countries and is diagnosed by the clinical appearance of yellow subretinal deposits called drusen. Genetic changes in immune components are clearly implicated in the pathology of this disease. We have previously shown that the purinergic receptor P2X7 can act as a scavenger receptor, mediating phagocytosis of apoptotic cells and insoluble debris. We performed a genetic association study of functional polymorphisms in the P2RX7 and P2RX4 genes in a cohort of 744 patients with AMD and 557 age-matched Caucasian control subjects. The P2X4 Tyr315Cys variant was 2-fold more frequent in patients with AMD compared to control subjects, with the minor allele predicting susceptibility to disease. Pairwise linkage disequilibrium was observed between Tyr315Cys in the P2RX4 gene and Gly150Arg in the P2RX7 gene, and these two minor alleles formed a rare haplotype that was overrepresented in patients with AMD (n=17) compared with control subjects (n=3) (odds ratio 4.05, P=0.026). Expression of P2X7 (wild type or variant 150Arg) in HEK293 cells conferred robust phagocytosis toward latex beads, whereas coexpression of the P2X7 150Arg with P2X4 315Cys variants almost completely inhibited phagocytic capacity. Fresh human monocytes harboring this heterozygous 150Arg-315Cys haplotype showed 40% reduction in bead phagocytosis. In the primate eye, immunohistochemistry indicated that P2X7 and P2X4 receptors were coexpressed on microglia and macrophages, but neither receptor was seen on retinal pigment epithelial cells. These results demonstrate that a haplotype including two rare variants in P2RX7 and P2RX4 confers a functional interaction between these two variant receptors that impairs the normal scavenger function of macrophages and microglia. Failure of this P2X7-mediated phagocytic pathway may impair removal of subretinal deposits and predispose individuals toward AMD.
Subject(s)
Genetic Predisposition to Disease/genetics , Macular Degeneration/genetics , Phagocytosis/genetics , Receptors, Purinergic P2X4/genetics , Receptors, Purinergic P2X7/genetics , Aged , Aged, 80 and over , Aging/genetics , Alleles , Female , Genetic Association Studies , Genotype , Haplotypes/genetics , Humans , Linkage Disequilibrium/genetics , Macular Degeneration/metabolism , Male , Middle Aged , Polymorphism, Single Nucleotide/genetics , Risk Factors , White People/geneticsABSTRACT
The P2X7 receptor gene (P2RX7) is highly polymorphic with five previously described loss-of-function (LOF) single-nucleotide polymorphisms (SNP; c.151+1G>T, c.946G>A, c.1096C>G, c.1513A>C and c.1729T>A) and one gain-of-function SNP (c.489C>T). The purpose of this study was to determine whether the functional P2RX7 SNPs are associated with lumbar spine (LS) bone mineral density (BMD), a key determinant of vertebral fracture risk, in post-menopausal women. We genotyped 506 post-menopausal women from the Aberdeen Prospective Osteoporosis Screening Study (APOSS) for the above SNPs. Lumbar spine BMD was measured at baseline and at 6-7 year follow-up. P2RX7 genotyping was performed by homogeneous mass extension. We found association of c.946A (p.Arg307Gln) with lower LS-BMD at baseline (P=0.004, ß=-0.12) and follow-up (P=0.002, ß=-0.13). Further analysis showed that a combined group of subjects who had LOF SNPs (n=48) had nearly ninefold greater annualised percent change in LS-BMD than subjects who were wild type at the six SNP positions (n=84; rate of loss=-0.94%/year and -0.11%/year, respectively, P=0.0005, unpaired t-test). This is the first report that describes association of the c.946A (p.Arg307Gln) LOF SNP with low LS-BMD, and that other LOF SNPs, which result in reduced or no function of the P2X7 receptor, may contribute to accelerated bone loss. Certain polymorphic variants of P2RX7 may identify women at greater risk of developing osteoporosis.
Subject(s)
Bone Density/genetics , Osteoporosis, Postmenopausal/genetics , Polymorphism, Single Nucleotide , Receptors, Purinergic P2X7/genetics , Spine/physiopathology , Female , Humans , Lumbosacral Region , Middle Aged , Osteoporosis, Postmenopausal/physiopathologyABSTRACT
The purinergic P2X7 receptor has a major role in the regulation of osteoblast and osteoclast activity and changes in receptor function may therefore affect bone mass in vivo. The aim of this study was to determine the association of non-synonymous single-nucleotide polymorphisms in the P2RX7 gene to bone mass and fracture incidence in post-menopausal women. A total of 1694 women (aged 45-58) participating in the Danish Osteoporosis Prevention Study were genotyped for 12 functional P2X7 receptor variants. Bone mineral density was determined at baseline and after 10 years. In addition, vertebral fracture incidence was documented at 10 years. We found that the rate of bone loss was clearly associated with the Arg307Gln amino acid substitution such that individuals heterozygous for this polymorphism had a 40% increased rate of bone loss. Furthermore, individuals carrying the Ile568Asn variant allele had increased bone loss. In contrast, the Gln460Arg polymorphism was associated with protection against bone loss. The Ala348Thr polymorphism was associated with a lower vertebral fracture incidence 10 years after menopause. Finally, we developed a risk model, which integrated P2RX7 genotypes. Using this model, we found a clear association between the low-risk (high-P2X7 function) alleles and low rate of bone loss. Conversely, high-risk (reduced P2X7 function) alleles were associated with a high rate of bone loss. In conclusion, an association was demonstrated between variants that reduce P2X7 receptor function and increased rate of bone loss. These data support that the P2X7 receptor is important in regulation of bone mass.
Subject(s)
Osteoporosis, Postmenopausal/genetics , Polymorphism, Single Nucleotide , Postmenopause/genetics , Receptors, Purinergic P2X7/genetics , Spinal Fractures/genetics , Bone Density/genetics , Female , Genetic Predisposition to Disease , Genotype , Humans , Middle Aged , Osteoporosis, Postmenopausal/complications , Spinal Fractures/etiology , Spinal Fractures/metabolismABSTRACT
The P2X4 receptor is involved in endothelium-dependent changes in large arterial tone in response to shear stress and is, therefore, potentially relevant to arterial compliance and pulse pressure. Four identified nonsynonymous polymorphisms in P2RX4 were reproduced in recombinantly expressed human P2X4. Electrophysiological studies showed that one of these, the Tyr315>Cys mutation (rs28360472), significantly reduced the peak amplitude of the ATP-induced inward current to 10.9% of wild-type P2X4 receptors in transfected HEK-293 cells (10 µmol/L of ATP; n=4-8 cells; P<0.001). Concentration-response curves for ATP and the partial agonist BzATP demonstrate that the 315Cys-P2X4 mutant had an increased EC(50) value for both ligands. Mutation of Tyr315>Cys likely disrupts the agonist binding site of P2X4 receptors, a finding supported by molecular modeling based on the zebrafish P2X4 receptor crystal structure. We tested inheritance of rs28360472 encoding the Tyr315>Cys mutation in P2RX4 against pulse pressure in 2874 subjects from the Victorian Family Heart Study. The minor allele frequency was 0.014 (1.4%). In a variance components analysis we found significant association with pulse pressure (P=0.023 for total association) where 1 minor allele increased pulse pressure by 2.84 mm Hg (95% CI: 0.41-5.27). This increase in pulse pressure associated with inheritance of 315Cys-P2X4 receptors might reflect reduced large arterial compliance as a result of impaired endothelium-dependent vasodilation in large arteries.