Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility

Ankylosing spondylitis is a common form of inflammatory arthritis predominantly affecting the spine and pelvis that occurs in approximately 5 out of 1,000 adults of European descent. Here we report the identification of three variants in the RUNX3, LTBR-TNFRSF1A and IL12B regions convincingly associated with ankylosing spondylitis (P < 5 × 10-8 in the combined discovery and replication datasets) and a further four loci at PTGER4, TBKBP1, ANTXR2 and CARD9 that show strong association across all our datasets (P < 5 × 10-6 overall, with support in each of the three datasets studied). We also show that polymorphisms of ERAP1, which encodes an endoplasmic reticulum aminopeptidase involved in peptide trimming before HLA class I presentation, only affect ankylosing spondylitis risk in HLA-B27-positive individuals. These findings provide strong evidence that HLA-B27 operates in ankylosing spondylitis through a mechanism involving aberrant processing of antigenic peptides.

Genomics of ankylosing spondylitis

Ankylosing spondylitis (AS) is the prototypic and most prevalent and debilitating spondyloarthropathy, a group of arthritides where the spine and pelvis are specifically targeted. Unlike many other forms of arthritis in which joint damage is mediated through tissue destruction, in AS uncontrolled bone formation occurs, frequently resulting in joint fusion and consequently significant disability. It is estimated that there are 2.4 million spondyloarthritis sufferers in the U.S., twice as many as rheumatoid arthritis. The pathogenesis of AS is very poorly understood and both genetics and gene expression profiling approaches have been utilized to elucidate the underlying mechanisms and pathways that drive the disease. Using powerful genome-wide association study approaches a number of candidate genes have been found to be associated with AS. However, although such approaches can identify genes that can contribute to the disease process, they do not inform us of the actual changes in gene/cell activity at any point in the disease process. Expression profiling allows us to take a "snapshot" of cellular activity and what gene activity changes are underlying those changes. A number of expression profiling studies have been undertaken in AS, looking at both circulating cells and tissues from affected joints. The results to date have been somewhat disappointing with little consensus on gene activity changes due to the low power of the studies undertaken. Some more recent better powered studies have identified diagnostic expression profiles that do point to a possible role for expression profiling in early AS diagnosis. Future studies will require collaborative approaches to target specific disease stages and sites with larger numbers of samples.

Genome-wide meta-analysis identifies 56 bone mineral density loci and reveals 14 loci associated with risk of fracture

Bone mineral density (BMD) is the most widely used predictor of fracture risk. We performed the largest meta-analysis to date on lumbar spine and femoral neck BMD, including 17 genome-wide association studies and 32,961 individuals of European and east Asian ancestry. We tested the top BMD-associated markers for replication in 50,933 independent subjects and for association with risk of low-trauma fracture in 31,016 individuals with a history of fracture (cases) and 102,444 controls. We identified 56 loci (32 new) associated with BMD at genome-wide significance (P < 5 × 10−8). Several of these factors cluster within the RANK-RANKL-OPG, mesenchymal stem cell differentiation, endochondral ossification and Wnt signaling pathways. However, we also discovered loci that were localized to genes not known to have a role in bone biology. Fourteen BMD-associated loci were also associated with fracture risk (P < 5 × 10−4, Bonferroni corrected), of which six reached P < 5 × 10−8, including at 18p11.21 (FAM210A), 7q21.3 (SLC25A13), 11q13.2 (LRP5), 4q22.1 (MEPE), 2p16.2 (SPTBN1) and 10q21.1 (DKK1). These findings shed light on the genetic architecture and pathophysiological mechanisms underlying BMD variation and fracture susceptibility.

Common variants in the region around Osterix are associated with bone mineral density and growth in childhood

Peak bone mass achieved in adolescence is a determinant of bone mass in later life. In order to identify genetic variants affecting bone mineral density (BMD), we performed a genome-wide association study of BMD and related traits in 1518 children from the Avon Longitudinal Study of Parents and Children (ALSPAC). We compared results with a scan of 134 adults with high or low hip BMD. We identified associations with BMD in an area of chromosome 12 containing the Osterix (SP7) locus, a transcription factor responsible for regulating osteoblast differentiation (ALSPAC: P = 5.8 × 10-4; Australia: P = 3.7 × 10-4). This region has previously shown evidence of association with adult hip and lumbar spine BMD in an Icelandic population, as well as nominal association in a UK population. A meta-analysis of these existing studies revealed strong association between SNPs in the Osterix region and adult lumbar spine BMD (P = 9.9 × 10-11). In light of these findings, we genotyped a further 3692 individuals from ALSPAC who had whole body BMD and confirmed the association in children as well (P = 5.4 × 10-5). Moreover, all SNPs were related to height in ALSPAC children, but not weight or body mass index, and when height was included as a covariate in the regression equation, the association with total body BMD was attenuated. We conclude that genetic variants in the region of Osterix are associated with BMD in children and adults probably through primary effects on growth.

Evidence-based recommendations for the diagnosis of ankylosing spondylitis: Results from the Australian 3E initiative in rheumatology

• As part of the 3E program, we conducted a systematic literature review and gathered consensus from 23 practising Australian rheumatologists to develop guidelines for early identification of ankylosing spondylitis and specialist referral. • In three rounds of break-out sessions followed by discussion and voting, the specialist panel addressed three questions related to diagnosis of ankylosing spondylitis: In individuals with back pain, what are the early clinical features that suggest ankylosing spondylitis? How useful is imaging in identifying early ankylosing spondylitis? Based on which clinical features should a general practitioner refer a patient to a rheumatologist for further evaluation? • The panel agreed on six recommendations related to the three questions: 1a. Early clinical features to suggest ankylosing spondylitis include inflammatory back pain and age at symptom onset < 45 years. 1b. The absence of symptomatic response to an appropriate course of non-steroidal anti-inflammatory drugs makes the diagnosis of ankylosing spondylitis less likely. 1c. Raised inflammatory markers are supportive, but their absence does not rule out the diagnosis of ankylosing spondylitis. 2a. Despite low sensitivity to detect changes of early ankylosing spondylitis, plain radiographs of the pelvis and spine are appropriate initial imaging techniques. 2b. Magnetic resonance imaging is a useful imaging modality for detecting early changes of ankylosing spondylitis. 3. Individuals with inflammatory back pain should be referred to a rheumatologist for further evaluation. • Effective dissemination and implementation of these recommendations are important to standardise the approach to early diagnosis of ankylosing spondylitis.

Influence of LRP5 polymorphisms on normal variation in BMD

Genetic studies based on cohorts with rare and extreme bone phenotypes have shown that the LRP5 gene is an important genetic modulator of BMD. Using family-based and case-control approaches, this study examines the role of the LRP5 gene in determining normal population variation of BMD and describes significant association and suggestive linkage between LRP5 gene polymorphisms and BMD in >900 individuals with a broad range of BMD. Introduction: Osteoporosis is a common, highly heritable condition determined by complex interactions of genetic and environmental etiologies. Genetic factors alone can account for 50-80% of the interindividual variation in BMD. Mutations in the LRP5 gene on chromosome 11q12-13 have been associated with rare syndromes characterized by extremely low or high BMD, but little is known about the contribution of this gene to the development of osteoporosis and determination of BMD in a normal population. Materials and Methods: To examine the entire spectrum of low to high BMD, 152 osteoporotic probands, their families (597 individuals), and 160 women with elevated BMD (T score > 2.5) were recruited. BMD at the lumbar spine, femoral neck, and hip were measured in each subject using DXA. Results: PAGE sequencing of the LRP5 gene revealed 10 single nucleotide polymorphisms (SNPs), 8 of which had allele frequencies of >5%, in exons 8, 9, 10, 15, and 18 and in introns 6, 7, and 21. Within families, a strong association was observed between an SNP at nucleotide C171346A in intron 21 and total hip BMD (p < 1 × 10-5 in men only, p = 0.0019 in both men and women). This association was also observed in comparisons of osteoporotic probands and unrelated elevated BMD in women (p = 0.03), along with associations with markers in exons 8 (C135242T, p = 0.007) and 9 (C141759T, p = 0.02). Haplotypes composed of two to three of the SNPs G121513A, C135242T, G138351A, and C141759T were strongly associated with BMD when comparing osteoporotic probands and high BMD cases (p < 0.003). An SNP at nucleotide C165215T in exon 18 was linked to BMD at the lumbar spine, femoral neck, and total hip (parametric LOD scores = 2.8, 2.5, and 2.2 and nonparametric LOD scores = 0.3, 1.1, and 2.2, respectively) but was not genetically associated with BMD variation. Conclusion: These results show that common LRP5 polymorphisms contribute to the determination of BMD in the general population.

Site and gender specificity of inheritance of bone mineral density

Differences in genetic control of BMD by skeletal sites and genders were examined by complex segregation analysis in 816 members of 147 families with probands with extreme low BMD. Spine BMD correlated more strongly in male-male comparisons and hip BMD in female-female comparisons, consistent with gender- and site-specificity of BMD heritability. Introduction: Evidence from studies in animals and humans suggests that the genetic control of bone mineral density (BMD) may differ at different skeletal sites and between genders. This question has important implications for the design and interpretation of genetic studies of osteoporosis. Methods: We examined the genetic profile of 147 families with 816 individuals recruited through probands with extreme low BMD (T-score < −2.5, Z-score < −2.0). Complex segregation analysis was performed using the Pedigree Analysis Package. BMD was measured by DXA at both lumbar spine (L1-L4) and femoral neck. Results: Complex segregation analysis excluded purely monogenic and environmental models of segregation of lumbar spine and femoral neck BMD in these families. Pure polygenic models were excluded at the lumbar spine when menopausal status was considered as a covariate, but not at the femoral neck. Mendelian models with a residual polygenic component were not excluded. These models were consistent with the presence of a rare Mendelian genotype of prevalence 3–19 %, causing high BMD at the hip and spine in these families, with additional polygenic effects. Total heritability range at the lumbar spine was 61–67 % and at the femoral neck was 44–67 %. Significant differences in correlation of femoral neck and lumbar spine BMD were observed between male and female relative pairs, with male-male comparisons exhibiting stronger lumbar spine BMD correlation than femoral neck, and female-female comparisons having greater femoral neck BMD correlation than lumbar spine. These findings remained true for parent-offspring correlations when menopausal status was taken into account. The recurrence risk ratio for siblings of probands of a Z-score < −2.0 was 5.4 at the lumbar spine and 5.9 at the femoral neck. Conclusions: These findings support gender- and site-specificity of the inheritance of BMD. These results should be considered in the design and interpretation of genetic studies of osteoporosis.

Genetic studies of disorders of calcium crystal deposition

In summary, although many factors are likely to be involved in regulating calcification and ossification processes, studies of the causation of articular chondrocalcinosis and disorders of spinal ossification, such as DISH and OPLL, implicate control over inorganic pyrophosphate levels as being one of the most important factors in their aetiopathogenesis. The findings of these studies may prove relevant to other rheumatic diseases in which ectopic ossification occurs, such as AS.

Genetic control of bone density and turnover: Role of the collagen 1α1, estrogen receptor, and vitamin D receptor genes

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1α1 (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of "s" alleles lost more bone than "SS" homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.

Suggestive linkage of the parathyroid receptor type 1 to osteoporosis

We have investigated the role of 23 candidate genes in the control of bone mineral density (BMD) by linkage studies in families of probands with osteoporosis (lumbar spine [LS] or femoral neck [FN] BMD T score < -2.5) and low BMD relative to an age- and gender-matched cohort (Z score < -2.0). One hundred and fifteen probands (35 male, 80 female) and 499 of their first- or second-degree relatives (223 males and 276 females) were recruited for the study. BMD was measured at the LS and FN using dual-energy X-ray absorptiometry and expressed as age- and gender-matched Z scores corrected for body mass index. The candidate genes studied were the androgen receptor, type I collagen A1 (COLIA1), COLIA2, COLIIA1, vitamin D receptor (VDR), colony-stimulating factor 1, calcium-sensing receptor, epidermal growth factor (EGF), estrogen receptor 1 (ESR1), fibrillin type 1, insulin-like growth factor 1, interleukin-1 alpha (IL-1α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), osteopontin, parathyroid hormone (PTH), PTH-related peptide, PTH receptor type 1 (PTHR1), transforming growth factor-beta 1, and tumor necrosis factors alpha and beta. Sixty-four microsatellites lying close to or within these genes were investigated for linkage with BMD. Using the program MapMaker/Sibs there was suggestive evidence of linkage between BMD and PTHR1 (maximum LOD score obtained [MLS] 2.7-3.5). Moderate evidence of linkage was also observed with EGF (MLS 1.8), COLIA1 (MLS 1.7), COLIIA1/VDR (MLS 1.7), ESR1 (MLS 1.4), IL-1α (MLS 1.4), IL-4 (MLS 1.2), and IL-6 (MLS 1.2). Variance components analysis using the program ACT, correcting for proband-wise ascertainment, also showed evidence of linkage (p ≤0.05) at markers close to or within the candidate genes IL- 1α, PTHR1, IL-6, and COLIIA1/VDR. Further studies will be required to confirm these findings, to refine the location of gene responsible for the observed linkage, and to screen the candidate genes targeted at these loci for mutations.

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.

3D numerical simulation of avascular tumour growth: effect of hypoxic micro-environment in host tissue

A three-dimensional (3D) mathematical model of tumour growth at the avascular phase and vessel remodelling in host tissues is proposed with emphasis on the study of the interactions of tumour growth and hypoxic micro-environment in host tissues. The hybrid based model includes the continuum part, such as the distributions of oxygen and vascular endothelial growth factors (VEGFs), and the discrete part of tumour cells (TCs) and blood vessel networks. The simulation shows the dynamic process of avascular tumour growth from a few initial cells to an equilibrium state with varied vessel networks. After a phase of rapidly increasing numbers of the TCs, more and more host vessels collapse due to the stress caused by the growing tumour. In addition, the consumption of oxygen expands with the enlarged tumour region. The study also discusses the effects of certain factors on tumour growth, including the density and configuration of preexisting vessel networks and the blood oxygen content. The model enables us to examine the relationship between early tumour growth and hypoxic micro-environment in host tissues, which can be useful for further applications, such as tumour metastasis and the initialization of tumour angiogenesis.

Tie-2 regulates the stemness and metastatic properties of prostate cancer cells

Ample evidence supports that prostate tumor metastasis originates from a rare population of cancer cells, known as cancer stem cells (CSCs). Unfortunately, little is known about the identity of these cells, making it difficult to target the metastatic prostate tumor. Here, for the first time, we report the identification of a rare population of prostate cancer cells that express the Tie-2 protein. We found that this Tie-2High population exists mainly in prostate cancer cell lines that are capable of metastasizing to the bone. These cells not only express a higher level of CSC markers but also demonstrate enhanced resistance to the chemotherapeutic drug Cabazitaxel. In addition, knockdown of the expression of the Tie-2 ligand angiopoietin (Ang-1) led to suppression of CSC markers, suggesting that the Ang-1/Tie-2 signaling pathway functions as an autocrine loop for the maintenance of prostate CSCs. More importantly, we found that Tie-2High prostate cancer cells are more adhesive than the Tie-2Low population to both osteoblasts and endothelial cells. Moreover, only the Tie-2High, but not the Tie-2Low cells developed tumor metastasis in vivo when injected at a low number. Taken together, our data suggest that Tie-2 may play an important role during the development of prostate tumor metastasis.

A genetic variant of MDM4 influences regulation by multiple microRNAs in prostate cancer

The oncogene MDM4, also known as MDMX or HDMX, contributes to cancer susceptibility and progression through its capacity to negatively regulate a range of genes with tumour-suppressive functions. As part of a recent genome-wide association study it was determined that the A-allele of the rs4245739 SNP (A>C), located in the 3'-UTR of MDM4, is associated with an increased risk of prostate cancer. Computational predictions revealed that the rs4245739 SNP is located within a predicted binding site for three microRNAs (miRNAs): miR-191-5p, miR-887 and miR-3669. Herein, we show using reporter gene assays and endogenous MDM4 expression analyses that miR-191-5p and miR-887 have a specific affinity for the rs4245739 SNP C-allele in prostate cancer. These miRNAs do not affect MDM4 mRNA levels, rather they inhibit its translation in C-allele-containing PC3 cells but not in LNCaP cells homozygous for the A-allele. By analysing gene expression datasets from patient cohorts, we found that MDM4 is associated with metastasis and prostate cancer progression and that targeting this gene with miR-191-5p or miR-887 decreases in PC3 cell viability. This study is the first, to our knowledge, to demonstrate regulation of the MDM4 rs4245739 SNP C-allele by two miRNAs in prostate cancer, and thereby to identify a mechanism by which the MDM4 rs4245739 SNP A-allele may be associated with an increased risk for prostate cancer.

Kallikrein-related peptidases in prostate cancer: From molecular function to clinical application

Prostate cancer is a leading contributor to male cancer-related deaths worldwide. Kallikrein-related peptidases (KLKs) are serine proteases that exhibit deregulated expression in prostate cancer, with KLK3, or prostate specific antigen (PSA), being the widely-employed clinical biomarker for prostate cancer. Other KLKs, such as KLK2, show promise as prostate cancer biomarkers and, additionally, their altered expression has been utilised for the design of KLK-targeted therapies. There is also a large body of in vitro and in vivo evidence supporting their role in cancer-related processes. Here, we review the literature on studies to date investigating the potential of other KLKs, in addition to PSA, as biomarkers and in therapeutic options, as well as their current known functional roles in cancer progression. Increased knowledge of these KLK-mediated functions, including degradation of the extracellular matrix, local invasion, cancer cell proliferation, interactions with fibroblasts, angiogenesis, migration, bone metastasis and tumour growth in vivo, may help define new roles as prognostic biomarkers and novel therapeutic targets for this cancer.