A direct comparison in diagnostic performance of CDUS, FDG-PET/CT and MRI in patients suspected of giant cell arteritis.

OBJECTIVES: This study directly compares diagnostic performance of Colour Duplex Ultrasound (CDUS), Fluor-18-deoxyglucose Positron Emission Tomography Computed Tomography (FDG-PET/CT) and Magnetic Resonance Imaging (MRI) in patients suspected of giant cell arteritis (GCA). METHODS: Patients with suspected GCA were included in a nested-case control pilot study. CDUS, whole body FDG-PET/CT and cranial MRI were performed within 5 working days after initial clinical evaluation. Clinical diagnosis after six months follow-up by experienced rheumatologists in the field of GCA, blinded for imaging, was used as reference standard. Diagnostic performance of the imaging modalities was determined. Stratification for GCA subtype was performed and imaging results were evaluated in different risk stratification groups. RESULTS: In total, 23 patients with GCA and 19 patients suspected of but not diagnosed with GCA were included. Sensitivity was 69.6% (95%CI 50.4%-88.8%) for CDUS, 52.2% (95%CI 31.4%-73.0%) for FDG-PET/CT and 56.5% (95%CI 35.8%-77.2%) for MRI. Specificity was 100% for CDUS, FDG-PET/CT and MRI. FDG-PET/CT was negative for GCA in all isolated cranial GCA patients (n = 8), while MRI was negative in all isolated extracranial GCA patients (n = 4). In 4 GCA patients with false-negative (n = 2; intermediate and high risk) or inconclusive (n = 2; low and intermediate risk) CDUS results, further imaging confirmed diagnosis. CONCLUSIONS: Sensitivity of CDUS was highest, while specificity was excellent in all imaging modalities. Nevertheless, confidence intervals of all imaging modalities were overlapping. Following EULAR recommendations, CDUS can be used as a first test to diagnose GCA. With insufficient evidence for GCA, further testing considering GCA subtype is warranted.

External validation of the giant cell arteritis probability score in the Netherlands.

OBJECTIVES: To prevent complications of giant cell arteritis (GCA), early and accurate diagnosis is essential. Recently, Laskou et al. (2019) developed the giant cell arteritis probability score (GCAPS) which allows physicians to assess the likelihood of GCA at an early stage. The aim of this study was to validate the GCAPS in a Dutch hospital. METHODS: A retrospective cohort of patients with suspected GCA between January 1st, 2017 and October 1st, 2019 was used. As the variable extracranial artery abnormality was not measured, a modified GCAPS was used (m-GCAPS). Clinical diagnosis of the rheumatologist after six months follow-up was used as reference. The m-GCAPS was assessed for discrimination and calibration. We applied risk stratification according to Sebastian et al. (2020) (low, intermediate and high-risk groups based on the median and 75th percentile). RESULTS: Our study included 209 suspected GCA patients. 135 patients had complete records. Of these patients, 40 had GCA. The m-GCAPS had an area under the curve of 0.83, a sensitivity of 80.0% and specificity of 75.8% at the optimal cut-off value >10.5. The Hosmer-Lemeshow test was non-significant. Using risk stratification, GCA prevalence was 12.5% in the low (score<9), 23.3% in the intermediate (9-12) and 78.6% in the high-risk group (>12). CONCLUSIONS: The m-GCAPS showed good discrimination and calibration in a Dutch retrospective cohort and can aid early recognition of GCA. Stratification into low, intermediate and high-risk is promising, but might need optimisation.

Diagnostic delay in patients with giant cell arteritis: results of a fast-track clinic.

Giant cell arteritis (GCA) can lead to severe complications if left untreated. The aim of this study was to describe time from onset of symptoms to diagnosis and treatment in GCA suspected patients in a fast-track clinic (FTC), and secondarily to assess the influence of GCA symptoms on this time. A retrospective cohort consisting of suspected GCA patients who visited the FTC between January 2017 and October 2019 was used. Time between symptom onset, first general practitioner visit, FTC referral, first FTC visit, and treatment initiation was analysed. Furthermore, this was stratified for subtypes of GCA and GCA symptoms. Of 205 patients referred with suspected GCA, 61 patients received a final diagnosis of GCA (GCA+) and 144 patients had no GCA (GCA-). Median time after onset of symptoms to first FTC visit was 31.0 days (IQR 13.0-108.8) in all referred patients. Time between onset of symptoms and first GP visit was 10.5 (4.0-36.3) days, and time between first GP visit and FTC referral was 10.0 (1.0-47.5) days. Patients were generally seen at the FTC within 1 day after referral. For patients with isolated cranial GCA (n = 41), median delay from onset of symptoms to treatment initiation was 21.0 days (11.0-73.5), while this was 57.0 days (33.0-105.0) in patients with extracranial large-vessel involvement (n = 20) (p = 0.02). Our results indicate considerable delay between symptom onset and FTC referral in patients suspected of GCA. Suspected patients were examined and GCA+ patients were treated instantly after referral. Key Points • GCA can cause severe complications with delayed treatment, but non-specific symptoms make diagnosis challenging. • Diagnostic delay still occurs despite introducing a successful fast-track clinic resulting from delay between start of symptoms and FTC referral. • Patients who presented with constitutional symptoms had longer delay than patients who presented with isolated cranial symptoms.

Comparing Diagnostic Performance of Short and Long [18F]FDG-PET Acquisition Times in Giant Cell Arteritis.

(1) Background: In giant cell arteritis (GCA), the assessment of cranial arteries using [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography (PET) combined with low-dose computed tomography (CT) may be challenging due to low image quality. This study aimed to investigate the effect of prolonged acquisition time on the diagnostic performance of [18F]FDG PET/CT in GCA. (2) Methods: Patients with suspected GCA underwent [18F]FDG-PET imaging with a short acquisition time (SAT) and long acquisition time (LAT). Two nuclear medicine physicians (NMPs) reported the presence or absence of GCA according to the overall image impression (gestalt) and total vascular score (TVS) of the cranial arteries. Inter-observer agreement and intra-observer agreement were assessed. (3) Results: In total, 38 patients were included, of whom 20 were diagnosed with GCA and 18 were without it. Sensitivity and specificity for GCA on SAT scans were 80% and 72%, respectively, for the first NMP, and 55% and 89% for the second NMP. On the LAT scans, these values were 65% and 83%, and 75% and 83%, respectively. When using the TVS, LAT scans showed especially increased specificity (94% for both NMPs). Observer agreement was higher on the LAT scans compared with that on the SAT scan. (4) Conclusions: LAT combined with the use of the TVS may decrease the number of false-positive assessments of [18F]FDG PET/CT. Additionally, LAT and TVS may increase both inter and intra-observer agreement.

TNF blockade requires 1,25(OH)2D3 to control human Th17-mediated synovial inflammation.

OBJECTIVES: T helper 17 (Th17) cells from patients with early rheumatoid arthritis (RA) induce a proinflammatory feedback loop upon RA synovial fibroblast (RASF) interaction, including autocrine interleukin (IL)-17A production. A major challenge in medicine is how to control the pathogenic Th17 cell activity in human inflammatory autoimmune diseases. The objective of this study was to examine whether tumour necrosis factor (TNF) blockade and/or 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) controls Th17-mediated synovial inflammation. METHODS: Peripheral CD4+CD45RO+CCR6+ Th17 cells of patients with early RA, Th17-RASF cocultures and synovial biopsy specimens were cultured with or without 1,25(OH)(2)D(3) and/or TNFα blockade. Intracellular cytokine expression was detected by flow cytometry. Cytokine and matrix metalloprotease (MMP) production was determined by ELISA. RESULTS: The authors show that the 1,25(OH)(2)D(3), but not TNFα blockade, significantly suppressed autocrine IL-17A production in Th17-RASF and synovial biopsy cultures. Combining 1,25(OH)(2)D(3) and TNFα blockade had a significant additive effect compared with single treatment in controlling synovial inflammation, indicated by a further reduction in IL-6, IL-8, MMP-1 and MMP-3 in Th17-RASF cocultures and IL-6 and IL-8 expression in cultures of RA synovial tissue. CONCLUSIONS: These data show that TNF blockade does not suppress IL-17A and IL-22, which can be overcome by 1,25(OH)(2)D(3). The combination of neutralising TNF activity and 1,25(OH)(2)D(3) controls human Th17 activity and additively inhibits synovial inflammation. This indicates more valuable therapeutic potential of activation of Vitamin D receptor signalling over current TNF neutralisation strategies in patients with RA and potentially other Th17-mediated inflammatory diseases.

Periarticular osteoporosis: a useful feature in the diagnosis of early rheumatoid arthritis? Reliability and validity in a cross-sectional diagnostic study using dual-energy X-ray absorptiometry.

OBJECTIVES: To identify regions of interest (ROIs) relevant to periarticular osteoporosis in RA with low precision error and sufficient inter-rater reliability and to test diagnostic validity for RA. METHODS: Periarticular BMD was measured using dual-energy X-ray absorptiometry (DXA). Five ROIs were defined around MCP and/or PIP joints II-V, II-IV and mid-metacarpal to mid-phalangeal. They were evaluated for precision using the root mean square coefficient of variation (RMS-CV) and the intra-class correlation coefficient (ICC) for inter-reader reliability. To test validity, established RA patients (n = 25) and early arthritis patients (n = 25) were compared with healthy controls (n = 37) matched on sex, age and menopausal status using paired t-tests, ROC curves and scatterplots. RESULTS: The RMS-CV was 0.45-1.07%. The ICC was 0.99. Mean BMDs of the five ROIs ranged from 0.321 to 0.372 g/cm(2) in established RA, from 0.321 to 0.382 g/cm(2) in early arthritis and from 0.342 to 0.401 g/cm(2) in healthy controls. Mean differences ranged from 0.012 to 0.032 g/cm(2) for established RA and from 0.023 to 0.033 g/cm(2) for early arthritis patients compared with matched controls, with P < 0.05 for ROIs 1-5 in early arthritis and the whole hand in established RA. ROC curves indicated low discriminative power, with an area under the curve (AUC) of 0.61-0.64, and scatterplots showed great overlap between BMD values of patients and controls. CONCLUSIONS: Periarticular BMD measured with DXA seems not to be a useful diagnostic feature due to strong overlap of BMD values between healthy controls, established RA patients and early arthritis patients.

The heterogeneous human memory CCR6+ T helper-17 populations differ in T-bet and cytokine expression but all activate synovial fibroblasts in an IFNγ-independent manner.

BACKGROUND: Chronic synovial inflammation is an important hallmark of inflammatory arthritis, but the cells and mechanisms involved are incompletely understood. Previously, we have shown that CCR6+ memory T-helper (memTh) cells and synovial fibroblasts (SF) activate each other in a pro-inflammatory feedforward loop, which potentially drives persistent synovial inflammation in inflammatory arthritis. However, the CCR6+ memTh cells are a heterogeneous population, containing Th17/Th22 and Th17.1 cells. Currently, it is unclear which of these subpopulations drive SF activation and how they should be targeted. In this study, we examined the individual contribution of these CCR6+ memTh subpopulations to SF activation and examined ways to regulate their function. METHODS: Th17/Th22 (CXCR3-CCR4+), Th17.1 (CXCR3+CCR4-), DP (CXCR3+CCR4+), and DN (CXCR3-CCR4-) CCR6+ memTh, cells sorted from PBMC of healthy donors or treatment-naïve early rheumatoid arthritis (RA) patients, were cocultured with SF from RA patients with or without anti-IL17A, anti-IFNγ, or 1,25(OH)2D3. Cultures were analyzed by RT-PCR, ELISA, or flow cytometry. RESULTS: Th17/Th22, Th17.1, DP, and DN cells equally express RORC but differ in production of TBX21 and cytokines like IL-17A and IFNγ. Despite these differences, all the individual CCR6+ memTh subpopulations, both from healthy individuals and RA patients, were more potent in activating SF than the classical Th1 cells. SF activation was partially inhibited by blocking IL-17A, but not by inhibiting IFNγ or TBX21. However, active vitamin D inhibited the pathogenicity of all subpopulations leading to suppression of SF activation. CONCLUSIONS: Human CCR6+ memTh cells contain several subpopulations that equally express RORC but differ in TBX21, IFNγ, and IL-17A expression. All individual Th17 subpopulations are more potent in activating SF than classical Th1 cells in an IFNγ-independent manner. Furthermore, our data suggest that IL-17A is not dominant in this T cell-SF activation loop but that a multiple T cell cytokine inhibitor, such as 1,25(OH)2D3, is able to suppress CCR6+ memTh subpopulation-driven SF activation.

Serum vitamin D levels are associated with structural and functional properties of the carotid artery in older men and women.

BACKGROUND AND AIMS: This cross-sectional study aimed to assess the relationship between serum vitamin D levels and carotid and brachial artery distensibility in patients older than 55 years, referred to the outpatient clinic of the department of internal medicine and geriatric medicine of the Erasmus Medical Center, in Rotterdam. METHODS AND RESULTS: From April to July 2006 we consecutively enrolled 49 elder patients (21 men and 28 women, mean age 78 ± 8 years) without a cardiovascular event within 6 weeks before the visit. Carotid and brachial artery distensibility coefficients and serum 25(OH)D levels (mean 50 ± 28.8 nmol/L) were assessed. Multivariate analysis (with linear regression model) was performed to investigate the relation between these parameters: carotid artery distensibility coefficient was associated with serum 25(OH)D levels (ß = 0.112; 95% CI 0.053 0.172; p = 0.001). Moreover, a negative association was also observed between carotid artery distensibility coefficient and mean arterial pressure (ß = -0.279; 95% CI, -0.339 -0.159; p = 0.0001). On the contrary, brachial artery distensibility has shown no association with 25(OH)D levels, being negatively linked to LDL-cholesterol levels and heart rate. An association was also observed between serum 25(OH)D level and carotid artery intima-media thickness. CONCLUSION: Our results revealed that serum 25(OH)D levels of older men and women were associated with both structural and functional properties of the carotid artery. No association was found with the brachial artery distensibility.

Ageing and vitamin D deficiency: effects on calcium homeostasis and considerations for vitamin D supplementation.

Vitamin D is a fat-soluble, seco-steroid hormone. In man, the vitamin D receptor is expressed in almost all tissues, enabling effects in multiple systems of the human body. These effects can be endocrine, paracrine and autocrine. The present review summarises the effects of ageing on the vitamin D endocrine system and on Ca homeostasis. Furthermore, consequences for vitamin D supplementation are discussed.

Human Memory Th17 Cell Populations Change Into Anti-inflammatory Cells With Regulatory Capacity Upon Exposure to Active Vitamin D.

Autoimmune diseases are characterized by an aberrantly activated immune system, resulting in tissue damage and functional disability in patients. An important therapeutic goal is to restore the deregulated immunological balance between pro- and anti-inflammatory T cells. This imbalance is illustrated by elevated levels and activity of memory Th17 cell populations, such as Th17, Th1/Th17, and Th17.1 cells, in various autoimmune diseases. These cells are characterized by the chemokine receptor CCR6, RORC expression and production of IL-17A, IFNγ, and TNFα. Using rheumatoid arthritis (RA) as a model of autoimmune disease, we here demonstrate that pro-inflammatory memory CCR6+ Th cells can switch into anti-inflammatory cells with regulatory capacity using the active vitamin D metabolite 1,25(OH)2D3. Memory CCR6+ Th cells, excluding Tregs, were sorted from healthy controls or treatment-naïve patients with early rheumatoid arthritis (RA) and cultured with or without 1,25(OH)2D3. Treatment with 1,25(OH)2D3 inhibited pro-inflammatory cytokines such as IL-17A, IL-17F, IL-22 and IFNγ in memory CCR6+ Th cells from both healthy controls and RA patients. This was accompanied by induction of anti-inflammatory factors, including IL-10 and CTLA4. Interestingly, these formerly pathogenic cells suppressed proliferation of autologous CD3+ T cells similar to classical Tregs. Importantly, the modulated memory cells still migrated toward inflammatory milieus in vitro, modeled by RA synovial fluid, and retained their suppressive capacity in this environment. These data show the potential to reset the pathogenic profile of human memory Th cells into non-pathogenic cells with regulatory capacity.

1,25(OH)2D3 and dexamethasone additively suppress synovial fibroblast activation by CCR6+ T helper memory cells and enhance the effect of tumor necrosis factor alpha blockade.

BACKGROUND: Despite recent improvements in the treatment of rheumatoid arthritis (RA), an insufficient treatment response and the development of treatment resistance in many patients illustrates the need for new therapeutic strategies. Chronic synovial inflammation could be suppressed by targeting RA synovial fibroblast (RASF) activation by, for example, interleukin (IL)-17A-producing CCR6+ T helper memory (memTh) cells. Here, we modulated this interaction by combining the active vitamin D metabolite 1,25(OH)2D3 with dexamethasone (DEX) and explored the potential therapeutic applications. METHODS: CCR6+ memTh cells from peripheral blood mononuclear cells (PBMCs) of healthy donors or treatment-naive early RA patients were cultured alone or with RASF from established RA patients for 3 days and treated with or without 1,25(OH)2D3, DEX, or etanercept. Treatment effects were assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. RESULTS: 1,25(OH)2D3, and to lesser extent DEX, reduced production of the pro-inflammatory cytokines IL-17A, IL-22, and interferon (IFN)γ in CCR6+ memTh cells. Tumor necrosis factor (TNF)α was only inhibited by the combination of 1,25(OH)2D3 and DEX. In contrast, DEX was the strongest inhibitor of IL-6, IL-8, and tissue-destructive enzymes in RASF. As a result, 1,25(OH)2D3 and DEX additively inhibited inflammatory mediators in CCR6+ memTh-RASF cocultures. Interestingly, low doses of mainly DEX, but also 1,25(OH)2D3, combined with etanercept better suppressed synovial inflammation in this coculture model compared with etanercept alone. CONCLUSION: This study suggests that 1,25(OH)2D3 and DEX additively inhibit synovial inflammation through targeting predominantly CCR6+ memTh cells and RASF, respectively. Furthermore, low doses of DEX and 1,25(OH)2D3 enhance the effect of TNFα blockade in inhibiting RASF activation, thus providing a basis to improve RA treatment.

Vitamin D in Autoimmunity: Molecular Mechanisms and Therapeutic Potential.

Over the last three decades, it has become clear that the role of vitamin D goes beyond the regulation of calcium homeostasis and bone health. An important extraskeletal effect of vitamin D is the modulation of the immune system. In the context of autoimmune diseases, this is illustrated by correlations of vitamin D status and genetic polymorphisms in the vitamin D receptor with the incidence and severity of the disease. These correlations warrant investigation into the potential use of vitamin D in the treatment of patients with autoimmune diseases. In recent years, several clinical trials have been performed to investigate the therapeutic value of vitamin D in multiple sclerosis, rheumatoid arthritis, Crohn's disease, type I diabetes, and systemic lupus erythematosus. Additionally, a second angle of investigation has focused on unraveling the molecular pathways used by vitamin D in order to find new potential therapeutic targets. This review will not only provide an overview of the clinical trials that have been performed but also discuss the current knowledge about the molecular mechanisms underlying the immunomodulatory effects of vitamin D and how these advances can be used in the treatment of autoimmune diseases.

Animal Models of Bone Loss in Inflammatory Arthritis: from Cytokines in the Bench to Novel Treatments for Bone Loss in the Bedside-a Comprehensive Review.

Throughout life, bone is continuously remodelled. Bone is formed by osteoblasts, from mesenchymal origin, while osteoclasts induce bone resorption. This process is tightly regulated. During inflammation, several growth factors and cytokines are increased inducing osteoclast differentiation and activation, and chronic inflammation is a condition that initiates systemic bone loss. Rheumatoid arthritis (RA) is a chronic inflammatory auto-immune disease that is characterised by active synovitis and is associated with early peri-articular bone loss. Peri-articular bone loss precedes focal bone erosions, which may progress to bone destruction and disability. The incidence of generalised osteoporosis is associated with the severity of arthritis in RA and increased osteoporotic vertebral and hip fracture risk. In this review, we will give an overview of different animal models of inflammatory arthritis related to RA with focus on bone erosion and involvement of pro-inflammatory cytokines. In addition, a humanised endochondral ossification model will be discussed, which can be used in a translational approach to answer osteoimmunological questions.

Stopping Tumor Necrosis Factor Inhibitor Treatment in Patients With Established Rheumatoid Arthritis in Remission or With Stable Low Disease Activity: A Pragmatic Multicenter, Open-Label Randomized Controlled Trial.

OBJECTIVE: Tumor necrosis factor inhibitor (TNFi) biologic agents are an effective treatment for rheumatoid arthritis (RA). It is unclear whether patients whose disease is in remission or who have stable low disease activity need to continue use of TNFi or can stop this treatment. This study was undertaken to assess whether patients with established RA who are in remission or have stable low disease activity can effectively and safely stop their TNFi therapy. METHODS: The study was designed as a pragmatic multicenter, open-label randomized controlled trial. Inclusion criteria were a diagnosis of RA according to the American College of Rheumatology 1987 classification criteria, as well as use of a TNFi for at least 1 year along with a stable dose of disease-modifying antirheumatic drugs and a Disease Activity Score in 28 joints (DAS28) of <3.2 over the 6 months preceding trial inclusion. Patients were randomized in a 2:1 ratio to either stop or continue treatment with their current TNFi. Flare was defined as a DAS28 of ≥3.2 during the 12-month follow-up period and an increase in score of ≥0.6 compared to the baseline DAS28. RESULTS: In total, 531 patients were allocated to the stop group and 286 to the TNFi continuation group. At 12 months, more patients had experienced a flare in the stop group (272 [51.2%] of 531) than in the continuation group (52 [18.2%] of 286; P < 0.001). The hazard ratio for occurrence of a flare after stopping TNFi was 3.50 (95% confidence interval [95% CI] 2.60-4.72). The mean DAS28 in the stop group was significantly higher during the follow-up period compared to that in the continuation group (P < 0.001). Of the 195 patients who restarted TNFi treatment after experiencing a flare and within 26 weeks after stopping, 165 (84.6%) had regained a DAS28 of <3.2 by 6 months later, and the median time to a regained DAS28 of <3.2 was 12 weeks (95% Cl 10.7-13.3). There were more hospitalizations in the stop group than in the continuation group (6.4% versus 2.4%). CONCLUSION: Stopping TNFi treatment results in substantially more flares than does continuation of TNFi in patients with established RA in remission or with stable low disease activity.

Emergency department visits due to vertebral fractures in the Netherlands, 1986-2008: steep increase in the oldest old, strong association with falls.

BACKGROUND: Vertebral fractures are a common consequence of osteoporosis in older persons. With the ageing of the population, numbers are expected to rise. OBJECTIVE: To determine trends in health care demand due to vertebral fracture related emergency department (ED) visits and hospitalizations in the older Dutch population. DESIGN AND SETTING: Secular trend analysis of vertebral fracture related ED visits between 1986 and 2008, using the Dutch Injury Surveillance System. All ED visits with a primary diagnosis of a vertebral fracture in persons aged ≥65 years were extracted from this database. MAIN OUTCOME MEASURE: Numbers, age-specific and age-adjusted incidence rates (per 100,000 population) of ED visits and hospitalization rates due to vertebral fractures in the older Dutch population were calculated for each year of the study. RESULTS: The total number of ED visits due to a vertebral fracture increased from 913 in 1986 to 2502 in 2008 (174% increase). The majority of fractures were caused by a low-energetic fall incident (83%). The overall age-adjusted incidence rate increased from 51.6 per 100,000 population in 1986 to 103.6 in 2008. Incidence rates increased with age and were higher in females than in males. The hospitalization rate remained stable at about 50-55%, in both females and males. CONCLUSION: Vertebral fracture related ED visits and hospitalizations are increasing rapidly in the older Dutch population, especially in the oldest-old. Most vertebral fractures were associated with falls. These findings indicate that a pro-active approach in the diagnosis and treatment of osteoporosis and in the prevention of falls in both men and women is warranted.

Association of 5' estrogen receptor alpha gene polymorphisms with bone mineral density, vertebral bone area and fracture risk.

This study investigates the influence of genetic variation of the estrogen receptor alpha (ESR1) gene locus on several bone parameters in 2042 individuals of The Rotterdam Study, a prospective population-based cohort study of elderly subjects. We analysed three polymorphic sites in the 5' region of the ESR1 gene; a (TA)(n)-repeat in the promoter region, and molecular haplotypes of the PvuII and XbaI RFLPs in intron 1, and inferred long-range haplotypes (LRH) thereof. We observed only three of the possible four PvuII-XbaI haplotypes in our population. A comparison with other Caucasian populations showed similar haplotype frequencies, while in Asian and African populations these were different. Linkage disequilibrium (LD) analysis between the PvuII-XbaI haplotype and the (TA)(n) repeat showed strong LD between the two sites. Reconstruction of long range haplotypes over the entire 5' region, revealed six frequent LRH. In men, we did not observe an association between the ESR1 polymorphisms studied and bone parameters. In women, we demonstrated an allele dose effect of haplotype "px" (P=0.003) and a low number of (TA)(n) repeats (P=0.008) with decreased lumbar spine bone mineral density (BMD) (4.8% lower BMD in women homozygous for haplotype "px", representing 28% of the population, compared with homozygous non-carriers) and decreased vertebral bone area (2.3% difference between extreme genotypes; P=0.016). Most importantly, we found an increased vertebral fracture risk with evidence for an allele dose effect with an odds ratio of 2.2 (95%CI 1.3-3.5) for haplotype "px", and 2.0 (1.5-3.2) for a low number of (TA)(n) repeats. The ESR1 genotype dependent fracture risk is largely independent of BMD and bone area. Combination of risk alleles at both loci by long-range haplotyping improved the associations slightly, but because of the strong LD between the two polymorphic sites, we were unable to determine if any particular polymorphic site is driving the associations found. We conclude that ESR1 polymorphism in the 5' (promoter) region is associated with vertebral fracture risk, lumbar spine BMD and vertebral bone area in postmenopausal women, but not in men. The molecular mechanism underlying this association needs further study.