Original article 555 Single nucleotide polymorphisms in the P2X 7 gene are associated to fracture risk and to effect of estrogen treatment Stine D. Ohlendorff a , Charlotte L. Tofteng a , Jens-Erik B. Jensen a , Solveig Petersen a , Roberto Civitelli e , Mogens Fenger a , Bo Abrahamsen b , Anne P. Hermann c , Pia Eiken d and Niklas R. Jørgensen a Objectives The purinergic P2RX 7 receptor (P2RX7) has been shown to play a role in the regulation of osteoblast and osteoclast activity. The aim of this study was to determine the presence of polymorphisms in exon 13 of the P2X 7 gene and the association with osteoclast apoptosis in vitro and bone status in vivo. Methods A total of 1764 postmenopausal women were genotyped for three single nucleotide polymorphisms detected after sequencing of exon 13 of P2X 7 . Bone markers, bone mineral density of the hip and lumbar spine were determined at baseline and after 10 years, and vertebral fracture incidence after 10 years. In-vitro ATP- induced caspase-1 determinations were performed on osteoclasts from the different genotypes. Results Three polymorphisms were detected (Gln460Arg, Glu496Ala, and Ile568Asn). None of the polymorphisms was related to bone mineral density or changes in bone mineral density over 10 years in hormone replacement therapy naı ¨ve women. The Ile568Asn polymorphism was however, associated with effect of hormone replacement therapy. Furthermore, the 10-year fracture incidence was significantly associated with both the Glu496Ala and the Ile568Asn. The Glu496Ala polymorphism was closely related to ATP-induced osteoclast apoptosis in vitro, as osteoclasts from individuals homozygous for the C allele had significantly decreased apoptotic activity. Conclusion The P2X 7 Glu496Ala and the Ile568Asn single nucleotide polymorphisms are associated with 10-year fracture risk in postmenopausal women and response to hormone replacement therapy treatment. Further, the Glu496Ala polymorphism is strongly influencing osteoclast apoptosis in vitro, which could contribute to increased fracture risk. Pharmacogenetics and Genomics 17: 555–567 c 2007 Lippincott Williams & Wilkins. Pharmacogenetics and Genomics 2007, 17:555–567 Keywords: fracture incidence, hormone replacement therapy, osteoclast, P2X 7 , polymorphism, purinergic a Departments of Endocrinology and Clinical Biochemistry, The Osteoporosis and Metabolic Research Unit, Copenhagen University Hospital Hvidovre, Hvidovre, b Department of Internal Medicine, Gentofte Hospital, Gentofte, c Department of Internal Medicine, Kolding Hospital, Kolding, d Department of Endocrinology, Hillerod Hospital, Hillerod, Denmark and e Division of Bone and Mineral Diseases, Department of Internal Medicine, Washington University, St Louis, Missouri, USA Correspondence to Niklas Rye Jørgensen, MD, PhD, DMSc, Department of Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Kettegaard Alle´ 30, DK-2650 Hvidovre, Denmark Tel: +45 36 32 23 65; fax: +45 36 32 36 40; e-mail: niklas@dadlnet.dk Received 29 June 2006 Accepted 9 January 2007 Introduction Osteoporosis is a disease that is related to occurrence of bone fractures and is pathophysiologically caused by loss of bone mass and deterioration of the microarchitectural structure of bone. Early diagnosis is essential in prevent- ing the disease, especially the occurrence of fractures. Mutations or polymorphisms of genes coding for proteins related to bone metabolism have been proposed as a means to predict future bone loss and eventually osteoporosis, and several studies have revealed that genetic factors play an important role in the regulation of bone mass [1]. Polymorphisms in genes for different bone-related proteins have been shown to be linked to changes in bone turnover and to fractures [2–5], but the effect size has been less than expected. Now consensus exists that osteoporosis is a multifactorial disease, caused by genetic, environmental, and metabolic factors, and that several genes are involved. Establishing an associa- tion between gene polymorphisms, bone mass, and bone turnover or fracture rates may indicate that a particular gene could be of importance for the pathogenesis of the disease. The purinergic P2X 7 receptor is a ligand-gated cation channel expressed primarily by cells of the hematopoietic lineage including macrophages [6,7] and osteoclasts [8– 10]. Ligand binding (ATP) to the receptor induces an opening of a channel permeable to small cations. Sustained activation, however, causes an increase in permeability (pore formation) by allowing bidirectional transport of a variety of ions and small molecules with a weight of up to 900 Da [11]. This effect is in certain cells associated with cytotoxicity. The physiological role of the P2X 7 receptor in osteoclasts is only vaguely elucidated, 1744-6872 c 2007 Lippincott Williams & Wilkins