Genetic studies in osteoporosis: The end of the beginning

Osteoporosis and disorders of bone fragility are highly heritable, but despite much effort the identities of few of the genes involved has been established. Recent developments in genetics such as genome-wide association studies are revolutionizing research in this field, and it is likely that further contributions will be made through application of next-generation sequencing technologies, analysis of copy number variation polymorphisms, and high-throughput mouse mutagenesis programs. This article outlines what we know about osteoporosis genetics to date and the probable future directions of research in this field.

Excessive bone formation in a mouse model of ankylosing spondylitis is associated with decreases in Wnt pathway inhibitors

Introduction: Ankylosing spondylitis (AS) is unique in its pathology where inflammation commences at the entheses before progressing to an osteoproliferative phenotype generating excessive bone formation that can result in joint fusion. The underlying mechanisms of this progression are poorly understood. Recent work has suggested that changes in Wnt signalling, a key bone regulatory pathway, may contribute to joint ankylosis in AS. Using the proteoglycan-induced spondylitis (PGISp) mouse model which displays spondylitis and eventual joint fusion following an initial inflammatory stimulus, we have characterised the structural and molecular changes that underlie disease progression. Methods: PGISp mice were characterised 12 weeks after initiation of inflammation using histology, immunohistochemistry (IHC) and expression profiling. Results: Inflammation initiated at the periphery of the intervertebral discs progressing to disc destruction followed by massively excessive cartilage and bone matrix formation, as demonstrated by toluidine blue staining and IHC for collagen type I and osteocalcin, leading to syndesmophyte formation. Expression levels of DKK1 and SOST, Wnt signalling inhibitors highly expressed in joints, were reduced by 49% and 63% respectively in the spine PGISp compared with control mice (P < 0.05) with SOST inhibition confirmed by IHC. Microarray profiling showed genes involved in inflammation and immune-regulation were altered. Further, a number of genes specifically involved in bone regulation including other members of the Wnt pathway were also dysregulated. Conclusions: This study implicates the Wnt pathway as a likely mediator of the mechanism by which inflammation induces bony ankylosis in spondyloarthritis, raising the potential that therapies targeting this pathway may be effective in preventing this process.

Whole blood transcriptional profiling in ankylosing spondylitis identifies novel candidate genes that might contribute to the inflammatory and tissue-destructive disease aspects

Introduction: A number of genetic-association studies have identified genes contributing to ankylosing spondylitis (AS) susceptibility but such approaches provide little information as to the gene activity changes occurring during the disease process. Transcriptional profiling generates a 'snapshot' of the sampled cells' activity and thus can provide insights into the molecular processes driving the disease process. We undertook a whole-genome microarray approach to identify candidate genes associated with AS and validated these gene-expression changes in a larger sample cohort. Methods: A total of 18 active AS patients, classified according to the New York criteria, and 18 gender- and age-matched controls were profiled using Illumina HT-12 whole-genome expression BeadChips which carry cDNAs for 48,000 genes and transcripts. Class comparison analysis identified a number of differentially expressed candidate genes. These candidate genes were then validated in a larger cohort using qPCR-based TaqMan low density arrays (TLDAs). Results: A total of 239 probes corresponding to 221 genes were identified as being significantly different between patients and controls with a P-value <0.0005 (80% confidence level of false discovery rate). Forty-seven genes were then selected for validation studies, using the TLDAs. Thirteen of these genes were validated in the second patient cohort with 12 downregulated 1.3- to 2-fold and only 1 upregulated (1.6-fold). Among a number of identified genes with well-documented inflammatory roles we also validated genes that might be of great interest to the understanding of AS progression such as SPOCK2 (osteonectin) and EP300, which modulate cartilage and bone metabolism. Conclusions: We have validated a gene expression signature for AS from whole blood and identified strong candidate genes that may play roles in both the inflammatory and joint destruction aspects of the disease.

The window of opportunity: A relevant concept for axial spondyloarthritis

The window of opportunity is a concept critical to rheumatoid arthritis treatment. Early treatment changes the outcome of rheumatoid arthritis treatment, in that response rates are higher with earlier disease-modifying anti-rheumatic drug treatment and damage is substantially reduced. Axial spondyloarthritis is an inflammatory axial disease encompassing both nonradiographic axial spondyloarthritis and established ankylosing spondylitis. In axial spondyloarthritis, studies of magnetic resonance imaging as well as tumor necrosis factor inhibitor treatment and withdrawal studies all suggest that early effective suppression of inflammation has the potential to reduce radiographic damage. This potential would suggest that the concept of a window of opportunity is relevant not only to rheumatoid arthritis but also to axial spondyloarthritis. The challenge now remains to identify high-risk patients early and to commence treatment without delay. Developments in risk stratification include new classification criteria, identification of clinical risk factors, biomarkers, genetic associations, potential antibody associations and an ankylosing spondylitis-specific microbiome signature. Further research needs to focus on the evidence for early intervention and the early identification of high-risk individuals.

Genetic studies of common rheumatological diseases

Editorial

Two monoclonal antibodies to precisely the same epitope of type ii collagen select non-crossreactive phage clones by phage display: Implications for autoimmunity and molecular mimicry

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.

Introduction: Epitope mimicry as a component cause of autoimmune disease

The causes of autoimmune diseases have yet to be fully elucidated. Autoantibodies, autoreactive T cell responses, the presence of a predisposing major histocompatibility complex (MHC) haplotype and responsiveness to corticosteroids are features, and some are possibly contributory causes of autoimmune disease. The most challenging question is how autoimmune diseases are triggered. Molecular mimicry of host cell determinants by epitopes of infectious agents with ensuing cross-reactivity is one of the most popular yet still controversial theories for the initiation of autoimmune diseases [1]. Throughout the 1990s, hundreds of research articles focusing to various extents on epitope mimicry, as it is more accurately described in an immunological context, were published annually. Many of these articles presented data that were consistent with the hypothesis of mimicry but that did not actually prove the theory. Other equally convincing reports indicated that epitope mimicry was not the cause of the autoimmune disease despite sequence similarity between molecules of infectious agents and the host. Some 20 years ago, Rothman [2] proposed a model for disease causation and I have used this as a framework to examine the role of epitope mimicry in the development of autoimmune disease. The thesis of Rothman’s model is that an effect, in this instance autoimmune disease, arises as a result of a cause. In most cases, multiple-component causes contribute synergistically to yield the effect, and each of these components alone is insufficient as a cause. Logically, some component causes, such as the presence of a particular autoimmune response, are also necessary causes.

Mimotopes identified by phage display for the monoclonal antibody CII- C1 to type II collagen

The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage. This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1). CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363. Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated. CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon. Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen. Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F. Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif ill the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive. These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen. There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA- 1. This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera. In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus.

Molecular mimicry: Can epitope mimicry induce autoimmune disease?

Mimicry of host antigens by infectious agents may induce cross-reactive autoimmune responses to epitopes within host proteins which, in susceptible individuals, may tip the balance of immunological response versus tolerance toward response and subsequently lead to autoimmune disease. Epitope mimicry may indeed be involved in the pathogenesis of several diseases such as post-viral myocarditis or Chagas disease, but for many other diseases in which it has been implicated, such as insulin-dependent diabetes mellitis or rheumatoid arthritis, convincing evidence is still lacking. Even if an epitope mimic can support a cross-reactive T or B cell response in vitro, its ability to induce an autoimmune disease in vivo will depend upon the appropriate presentation of the mimicked host antigen in the target tissue and, in the case of T cell mimics, the ability of the mimicking epitope to induce a proliferative rather than anergizing response upon engagement of the MHC-peptide complex with the T cell receptor. B cell presentation of mimicking foreign antigen to T cells is a possible mechanism for instigating an autoimmune response to self antigens that in turn can lead to autoimmune disease under particular conditions of antigen presentation, secondary signalling and effector cell repertoire. In this review evidence in support of epitope mimicry is examined in the light of the necessary immunological considerations of the theory.

Cuff tear arthropathy: Evidence of functional variation in pyrophosphate metabolism genes

We investigated the role of two genes, ANKH and TNAP, in patients with cuff tear arthropathy. These genes encode proteins which regulate the extracellular concentration of inorganic pyrophosphate, fluctuations of which can lead to calcium crystal formation. Variants were detected by direct sequencing of DNA and their frequencies compared with healthy controls. The effect of variants on protein function was further studied by in vitro approaches. Variant genotypes were observed more frequently in the cases when compared with controls in ANKH (45% and 20%) and TNAP (32% and 9%). Variants in ANKH altered inorganic pyrophosphate (PPi) concentrations in transfected human chondrocytes. There was a higher mean serum concentration of TNAP detected in female patients compared with normal ranges. Cuff tear arthropathy is associated with variants in ANKH and TNAP that alter extracellular inorganic pyrophosphate concentrations causing calcium crystal deposition. This supports a theory that genetic variants predispose patients to primary crystal deposition which when combined with a massive rotator cuff tear leads to the development of arthritis.

Predisposing factors to spondyloarthropathies

Predisposition to ankylosing spondylitis is largely genetic, and epidemiologic studies suggest that the environmental trigger is ubiquitous. HLA-B27 and -B60 predispose to ankylosing spondylitis, but in neither case is the mechanism of effect known. Other major histocompatibility complex and non-major histocompatibility complex genes are likely to influence susceptibility to spondyloarthritis as well as the disease pattern. Spondyloarthritis occurs in genetically predisposed inviduals exposed to certain as yet undefined environmental triggers. Although genes within the major histocompatibility complex are clearly major determinants of susceptibility to spondyloarthritis, epidemiologic evidence suggests that their contribution accounts for less than 50% of the total. The mechanism of association of B27 with these diseases is unknown; we are currently unable to predict which E27 carriers will develop arthritis or which form of BP27-associated spondyloarthritis they will develop. Lessons from transgenic animal experiments and technical and statistical advances in the field of genetics have greatly increased our ability to investigate these questions.

Transcutaneous bone-anchoring prosthesis with knee replacement: A novel treatment for amputees

Background Over the last two decades, Transcutaneous Bone-Anchored Prosthesis (TCBAP) has proven to be an effective alternative for prosthetic attachment for amputees, particularly for individuals unable to wear a socket. [1-17] However, the load transmitted through a typical TCBAP to the residual tibia and knee joint can be unbearable for transtibial amputees with knee arthritis. Aim A. To describe the surgical procedure combining TKR with TCBAP for the first time; and B. To present preliminary data on potential risks and benefits with assessment of clinical and functional outcomes at follow up Method We used a TCBAP connected to the tibial base plate of a Total Knee Replacement (TKR) prosthesis enabling the tibial residuum and the knee joint to act as weight sharing structures by transferring the load directly to the femur. We performed a standard hinged TKR connected to a custom made TCBAP at the first stage followed by creating a skin implant interface as a second stage. We retrospectively reviewed four cases of trans-tibial amputations presenting with knee joint arthritis. Patients were assessed clinically and functionally including standard measures of health-related quality of life, amputee mobility predictor tool, ambulation tests and actual activity level. Progress was monitored for 6-24 months. Results Clinical outcomes including adverse events show no major complications but one case of superficial infection. Functional outcomes improved for all participants as early as 6 months follow up. Discussion & Conclusion TKR and TCBAP were combined for the first time in this proof-of-concept case series. The preliminary outcomes indicated that this procedure is potentially a safe and effective alternative for this patient group despite the theoretical increase in risk of ascending infection through the skin-implant interface to the external environment. We suggest larger comparative series to further validate these results.

Cardiometabolic effects of genetic upregulation of the interleukin 1 receptor antagonist: A Mendelian randomisation analysis

Background To investigate potential cardiovascular and other effects of long-term pharmacological interleukin 1 (IL-1) inhibition, we studied genetic variants that produce inhibition of IL-1, a master regulator of inflammation. Methods We created a genetic score combining the effects of alleles of two common variants (rs6743376 and rs1542176) that are located upstream of IL1RN, the gene encoding the IL-1 receptor antagonist (IL-1Ra; an endogenous inhibitor of both IL-1α and IL-1β); both alleles increase soluble IL-1Ra protein concentration. We compared effects on inflammation biomarkers of this genetic score with those of anakinra, the recombinant form of IL-1Ra, which has previously been studied in randomised trials of rheumatoid arthritis and other inflammatory disorders. In primary analyses, we investigated the score in relation to rheumatoid arthritis and four cardiometabolic diseases (type 2 diabetes, coronary heart disease, ischaemic stroke, and abdominal aortic aneurysm; 453 411 total participants). In exploratory analyses, we studied the relation of the score to many disease traits and to 24 other disorders of proposed relevance to IL-1 signalling (746 171 total participants). Findings For each IL1RN minor allele inherited, serum concentrations of IL-1Ra increased by 0·22 SD (95% CI 0·18–0·25; 12·5%; p=9·3 × 10−33), concentrations of interleukin 6 decreased by 0·02 SD (−0·04 to −0·01; −1·7%; p=3·5 × 10−3), and concentrations of C-reactive protein decreased by 0·03 SD (−0·04 to −0·02; −3·4%; p=7·7 × 10−14). We noted the effects of the genetic score on these inflammation biomarkers to be directionally concordant with those of anakinra. The allele count of the genetic score had roughly log-linear, dose-dependent associations with both IL-1Ra concentration and risk of coronary heart disease. For people who carried four IL-1Ra-raising alleles, the odds ratio for coronary heart disease was 1·15 (1·08–1·22; p=1·8 × 10−6) compared with people who carried no IL-1Ra-raising alleles; the per-allele odds ratio for coronary heart disease was 1·03 (1·02–1·04; p=3·9 × 10−10). Per-allele odds ratios were 0·97 (0·95–0·99; p=9·9 × 10−4) for rheumatoid arthritis, 0·99 (0·97–1·01; p=0·47) for type 2 diabetes, 1·00 (0·98–1·02; p=0·92) for ischaemic stroke, and 1·08 (1·04–1·12; p=1·8 × 10−5) for abdominal aortic aneurysm. In exploratory analyses, we observed per-allele increases in concentrations of proatherogenic lipids, including LDL-cholesterol, but no clear evidence of association for blood pressure, glycaemic traits, or any of the 24 other disorders studied. Modelling suggested that the observed increase in LDL-cholesterol could account for about a third of the association observed between the genetic score and increased coronary risk. Interpretation Human genetic data suggest that long-term dual IL-1α/β inhibition could increase cardiovascular risk and, conversely, reduce the risk of development of rheumatoid arthritis. The cardiovascular risk might, in part, be mediated through an increase in proatherogenic lipid concentrations. Funding UK Medical Research Council, British Heart Foundation, UK National Institute for Health Research, National Institute for Health Research Cambridge Biomedical Research Centre, European Research Council, and European Commission Framework Programme 7.

Genetics of ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis that affects the spine and sacroiliac joints. It causes significant disability and is associated with a number of other features including peripheral arthritis, anterior uveitis, psoriasis and inflammatory bowel disease (IBD). Significant progress has been made in the genetics of AS have in the last five years, leading to new treatments in trial, and major leaps in understanding of the aetiopathogenesis of the disease.

Consensus statement on the investigation and management of non-radiographic axial spondyloarthritis (nr-axSpA)

Aim Non-radiographic axial spondyloarthritis (nr-axSpA) is axial inflammatory arthritis where plain radiographic damage is not evident. An unknown proportion of these patients will progress to ankylosing spondylitis (AS). The increasing recognition of nr-axSpA has been greatly assisted by the widespread use of magnetic resonance imaging. The aim of this article was to construct a set of consensus statements based on a literature review to guide investigation and promote best management of nr-axSpA. Methods A literature review using Medline was conducted covering the major investigation modalities and treatment options available. A group of rheumatologists and a radiologist with expertise in investigation and management of SpA reviewed the literature and formulated a set of consensus statements. The Grade system encompassing the level of evidence and strength of recommendation was used. The opinion of a patient with nr-axSpA and a nurse experienced in the care of SpA patients was also sought and included. Results The literature review found few studies specifically addressing nr-axSpA, or if these patients were included, their results were often not separately reported. Fourteen consensus statements covering investigation and management of nr-axSpA were formulated. The level of agreement was high and ranged from 8.1 to 9.8. Treatment recommendations vary little with established AS, but this is primarily due to the lack of available evidence on the specific treatment of nr-axSpA. Conclusion The consensus statements aim to improve the diagnosis and management of nr-axSpA. We aim to raise awareness of this condition by the public and doctors and promote appropriate investigation and management.