The role of LCN2 and LCN2-MMP9 in spondylitis radiographic development: gender and HLA-B27 status differences.

BACKGROUND: Male HLA-B27-positive radiographic-axial spondyloarthritis (r-axSpA) patients are prone to have severe spinal radiographic progression, but the underlying mechanisms are unclear. We recently showed that persistently elevated Lipocalin 2 (LCN2; L) reflects sacroiliac joint (SIJ) inflammation. LCN2 binds to MMP9. Concomitant elevation of L and LCN2-MMP9 (LM) was detected in many inflammatory diseases. We asked whether L and LM play similar roles in r-axSpA pathogenesis. METHODS: We analyzed 190 axSpA patients (123 radiographic and 67 non-radiographic axSpA) who had no detectable circulating Oncostatin M, to avoid complications due to cross-talk between pathways. L and LM levels from a single blood sample of each patient were measured and were correlated with MRI and modified stoke AS (mSASS) scoring. Association of elevated L (L+) or concurrent L+ and elevated LM (LM+) patterns with B27 status and gender were assessed. RESULTS: In L+LM+ axSpA patients, both L and LM levels correlated with MRI SPARCC SIJ scores, but only LM levels correlated with MRI Berlin Spine Scores, suggesting LM is a biomarker for both SIJ and spinal inflammation. Among patients with minimal spinal ankylosis (mSASSS < 10), 65% of male r-axSpA patients are L+LM+, while 30% and 64% of female patients are L+LM+ and L+, respectively, supporting the role of LM with disease progression. In B27+ L+LM+ male patients, both L and LM (but not CRP) levels correlate with mSASSS. B27 positivity and maleness have additive effects on spondylitis progression, suggesting concurrent high L and LM elevations are associated with B27+ male patients having more significant radiographic damage. L+ B27-negative male patients or L+ female patients are more likely to have milder disease. CONCLUSION: L and LM are informative biomarkers for SIJ and spinal inflammation, as well as for ankylosing development in r-axSpA patients. Distinctive L+LM+ or L+ patterns not only could distinguish clinically aggressive vs milder course of disease, respectively, but also provide an explanation for B27-positive male patients being the most susceptible to severe ankylosis.

Serial Lipocalin 2 and Oncostatin M levels reflect inflammation status and treatment response in axial spondyloarthritis.

BACKGROUND: Informative serum biomarkers for monitoring inflammatory activity and treatment responses in axial spondyloarthritis (axSpA) are lacking. We assessed whether Lipocalin 2 (LCN2) and Oncostatin M (OSM), both having roles in inflammation and bone remodeling, may accurately reflect chronic joint inflammation and treatment response in axSpA. Previous reports in animal models showed involvement of LCN2 and OSM in joint/gut inflammation. We asked whether they also play a role in human axSpA. METHODS: We analyzed a longitudinal observational axSpA cohort (286 patients) with yearly clinical assessments and concurrent measurements of serum LCN2 and OSM (1204 serum samples) for a mean of 4 years. Biomarker levels were correlated with MRI scoring and treatment response. RESULTS: Persistent and transient elevation of LCN2 and OSM were observed in axSpA patients. Persistent elevation of LCN2 or OSM, but not CRP, correlated with sacroiliac joint (SIJ) MRI SPARCC scores (Pearson's correlation p = 0.0005 and 0.005 for LCN2 and OSM respectively), suggesting that LCN2/OSM outperforms CRP as reflective of SIJ inflammation. We observed both concordant and discordant patterns of LCN2 and OSM in relationship to back pain, the cardinal clinical symptom in axSpA. Twenty-six percent (73/286) of the patients remained both clinically and serologically active (CASA). Sixty percent (173/286) of the patients became clinically quiescent, with back pain resolved, but 53% (92/173) of them were serologically active (CQSA), indicating that pain control may not indicate control of joint inflammation, as reflected by positive MRI imaging of SIJ. With respect to treatment responses, transient elevation of LCN2 or OSM over time was predictive of better response to all treatments. CONCLUSION: In axSpA, persistent LCN2 and/or OSM elevation reflects chronic SIJ inflammation and suboptimal treatment response. In our cohort, half of the currently deemed clinically quiescent patients with back pain resolved continued to demonstrate chronic joint inflammation. LCN2 and OSM profiling outperforms CRP as a predictive measure and provides an objective assessment of chronic local inflammation in axSpA patients.

Lipocalin 2 links inflammation and ankylosis in the clinical overlap of inflammatory bowel disease (IBD) and ankylosing spondylitis (AS).

BACKGROUND: Little is known about the mechanisms underlying the clinical overlap between gut inflammation and joint ankylosis, as exemplified by the concurrence of inflammatory bowel diseases (IBD) and ankylosing spondylitis (AS). As dysbiosis may serve as a common contributor, the anti-microbial pleiotropic factor lipocalin 2 could be a potential mediator due to its roles in inflammation and bone homeostasis. METHODS: Baseline colonic pathology was conducted in the ank/ank mouse model. Serum lipocalin 2 was analyzed by ELISA, in ank/ank mutants versus C3FeB6-A/Aw-jwt/wt, in patients with concurrent AS-IBD, AS alone, IBD alone, or mechanical back pain, and in healthy controls. In the ank/ank mouse model, the expression of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) was examined by real-time PCR. Intraperitoneal injection was done with the PPARγ agonist rosiglitazone or antagonist bisphenol A diglycidyl ether for four consecutive days. Serum levels of lipocalin 2 were examined on the sixth day. RESULTS: This study showed that the ank/ank mice with fully fused spines had concurrent colonic inflammation. By first using the ank/ank mouse model with progressive ankylosis and subclinical colonic inflammation, confirmed in patients with concurrent AS and IBD, elevated circulating lipocalin 2 levels were associated with the coexisting ankylosis and gut inflammation. The intracellular pathway of lipocalin 2 was further investigated with the ank/ank mouse model involving PPARγ. Colonic expression of PPARγ was negatively associated with the degree of gut inflammation. The PPARγ agonist rosiglitazone treatment significantly upregulated the serum levels of lipocalin 2, suggesting a potential regulatory role of PPARγ in the aberrant expression of lipocalin 2. CONCLUSIONS: In summary, lipocalin 2 modulated by PPARγ could be a potential pathway involved in concurrent inflammation and ankylosis in AS and IBD.

The genetic basis of ankylosing spondylitis: new insights into disease pathogenesis.

Ankylosing spondylitis (AS) is a complex disease involving multiple risk factors, both genetic and environmental. AS patients are predominantly young men, and the disease is characterized by inflammation and ankylosis, mainly at the cartilage-bone interface and enthesis. HLA-B27 has been known to be the major AS-susceptibility gene for more than 40 years. Despite advances made in the past few years, progress in the search for non-human leukocyte antigen susceptibility genes has been hampered by the heterogeneity of the disease. Compared to other complex diseases, such as inflammatory bowel disease (IBD), fewer susceptibility loci have been identified in AS. Furthermore, non-major histocompatibility-complex susceptibility loci discovered, such as ERAP1 and IL23R, are likely contributors to joint inflammation. Identification and confirmation of functional variants remains a significant challenge of investigations involving genome-wide association studies (GWAS). It remains unclear why none of the AS-susceptibility genes identified in GWAS appear to be directly involved in the ankylosing process. Numerous reviews have recently been published on the genetics of AS. Therefore, aside from a brief summary of what AS GWAS has successfully achieved thus far, this review will focus on directions that could address unanswered questions raised by GWAS.

Serum levels of novel noggin and sclerostin-immune complexes are elevated in ankylosing spondylitis.

BACKGROUND: Unravelling the basis of joint inflammation and ankylosis represents a major challenge in ankylosing spondylitis (AS) research. As noggin (NOG) and sclerostin (SOST) have recently been associated with the disease process in mouse and human studies, respectively, we explored the immune responses to these two molecules in AS. METHODS: Immune complexes (IC) composed of IgG autoantibodies to NOG and SOST were detected by immunoprecipitation and Western blot analyses. Epitope-specific IgG were measured using peptide-binding ELISA. Serum samples were obtained from healthy controls and patients with AS, mechanical back pain (MBP) and inflammatory bowel disease (IBD) with or without concomitant AS. RESULTS: NOG and SOST-IgG IC were present in NOG-treated and untreated ank/ank (progressive ankylosis), but not in wild-type mice. Higher than normal levels of NOG and SOST-IgG IC are present in AS sera (p<0.001). We showed a SOST peptide (SOST-S146, with homology to a bacterial glycotransferase peptide) binds to a NOG peptide (NOG-N54), which contains a N-glycosylation site. AS patients have higher levels of IgG recognising the NOG-N54 and SOST-S146 peptides compared to the levels in normal controls, IBD and MBP patients (one way analysis of variance p<0.0001). CONCLUSIONS: This is the first report showing IgG autoantibodies to NOG and SOST in normal individuals, and higher levels of NOG and/or SOST-IgG IC probably contribute to neo-ossification in AS patients. These novel findings hold the promise of earlier diagnosis, better management of AS with comorbidities and new therapeutic approaches to modulate ankylosis in AS.

Aberrant chondrocyte hypertrophy and activation of ß-catenin signaling precede joint ankylosis in ank/ank mice.

OBJECTIVE: We assessed the role of Ank in the maintenance of postnatal articular cartilage using the ank/ank mouse (mice homozygous for progressive ankylosis). METHODS: We analyzed ank/ank mice and wild-type littermates (8, 12, and 18 weeks old). Sections from decalcified, paraffin-embedded joints were stained with hematoxylin and eosin. Articular chondrocyte size and cartilage thickness were determined using morphometric methods. Immuno-histochemical staining was performed with anticollagen X, antitissue nonspecific alkaline phosphatase (TNAP), and anti-ß-catenin antibodies on fixed joint sections. Axin2 expression in paw joint lysates in wild-type versus ank/ank mice were compared using Western blot analysis. RESULTS: In all age groups of normal mice studied, calcified cartilage (CC) chondrocyte areas were significantly larger than those of uncalcified cartilage (UC) chondrocytes. However, similar chondrocyte areas (UC vs CC) were found in 12-week and 18-week-old ank/ank mice, indicating that hypertrophic chondrocytes were present in the UC of these mutant mice. The ank/ank mice showed an increase in CC thickness. The ank/ank UC hypertrophic chondrocytes showed diffuse immuno-reactivity for collagen X and TNAP. Increased ß-catenin activation was demonstrated by nuclear localization of ß-catenin staining in ank/ank chondrocytes. Axin2 expression from paw lysates was downregulated in ank/ank mice. CONCLUSION: We identified a previously unrecognized phenotype in the articular cartilage of ank/ank mice: collagen X-positive hypertrophic chondrocytes in the UC. It is possible that consequent to downregulation of axin2 expression, ß-catenin signaling was activated, leading to accelerated chondrocyte maturation and eventual ankylosis in ank/ank joints. Our studies shed new light on the contribution of a key signaling pathway in this model of joint ankylosis.

Aberrant axial mineralization precedes spinal ankylosis: a molecular imaging study in ank/ank mice.

INTRODUCTION: The diagnosis of ankylosing spondylitis is made from a combination of clinical features and the presence of radiographic evidence that may be detected only after many years of inflammatory back pain. It is not uncommon to have a diagnosis confirmed 5 to 10 years after the initial onset of symptoms. Development of a more-sensitive molecular imaging technology to detect structural changes in the joints would lead to earlier diagnosis and quantitative tracking of ankylosis progression. Progressive ankylosis (ank/ank) mice have a loss of function in the Ank gene, which codes for a regulator of PPi transport. In this study, we used these ank/ank mutant mice to assess a noninvasive, quantitative measure of joint ankylosis with near-infrared (NIR) molecular imaging in vivo. METHODS: Three age groups (8, 12, and 18 weeks) of ank/ank (15 mice) and wild-type littermates (12 +/+ mice) were assessed histologically and radiographically. Before imaging, OsteoSense 750 (bisphosphonate pamidronate) was injected i.v. Whole-body images were analyzed by using the multispectral Maestro imaging system. RESULTS: OsteoSense 750 signals in the paw joints were higher in ank/ank mice in all three age groups compared with controls. In the spine, significantly higher OsteoSense 750 signals were detected early, in 8-week-old ank/ank mice compared with controls, although minimal radiographic differences were noted at this time point. The molecular imaging changes in the ank/ank spine (8 weeks) were supported by histologic changes, including calcium apatite crystals at the edge of the vertebral bodies and new syndesmophyte formation. CONCLUSIONS: Changes in joint pathology of ank/ank mice, as evaluated by histologic and radiographic means, are qualitative, but only semiquantitative. In contrast, molecular imaging provides a quantitative assessment. Ankylosis in ank/ank mice developed simultaneously in distal and axial joints, contrary to the previous notion that it is a centripetal process. NIR imaging might be feasible for early disease diagnosis and for monitoring disease progression in ankylosing spondylitis.

Serum cytokine receptors in ankylosing spondylitis: relationship to inflammatory markers and endoplasmic reticulum aminopeptidase polymorphisms.

OBJECTIVE: Endoplasmic reticulum aminopeptidase (ERAP)1 is associated with ankylosing spondylitis (AS) and is known to be involved in the clipping of the cytokine receptors interleukin 1 receptor II (IL-1RII), IL-6Ralpha, and tumor necrosis factor receptor I (TNFRI). We studied the relationship of these serum cytokine receptors and their corresponding cytokines to markers of inflammation and polymorphisms in ERAP1 and ERAP2 in patients with AS. METHODS: Sera from patients with AS were assayed for TNF-alpha, IL-1, IL-6, sTNFRI, sIL-1RII, and sIL-6Ralpha by ELISA. Genotyping was performed for 3 AS-associated nonsynonymous single-nucleotide polymorphisms in the ERAP1 gene [rs27044(C/G), rs10050860(C/T), and rs30187(C/T)] and 1 in the ERAP2 gene [rs2549782(T/G)]. The serum cytokine and receptor levels were compared between the different genotype groups and correlated to markers of inflammation and disease activity. RESULTS: Eighty patients with AS (21 women) with a mean Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) of 5.3 +/- 2.4 were enrolled. There was a significant correlation of sTNFRI with C-reactive protein (CRP; R = 0.43, p < 0.001) and erythrocyte sedimentation rate (ESR; R = 0.30, p = 0.01) but not with BASDAI. Serum cytokine levels were undetectable in the majority of patients. There was no significant difference in serum cytokines or the soluble receptors between patients with the different ERAP1/ERAP2 polymorphisms and their haplotypes. Similarly, there was no relationship of the polymorphisms with the serum cytokine levels nor the cytokine-receptor ratio. CONCLUSION: Soluble TNFRI levels correlate with ESR and CRP in AS. The ERAP1 and ERAP2 polymorphisms associated with AS do not influence the serum cytokine receptor levels in patients with AS.

Association of an ERAP1 ERAP2 haplotype with familial ankylosing spondylitis.

OBJECTIVES: To assess whether there is excess transmission of alleles from the ERAP1 ERAP2 locus in families with ankylosing spondylitis (AS). METHODS: 199 multiplex families with AS with four non-synonymous single nucleotide polymorphisms (SNPs), three in the endoplasmic reticulum aminopeptidase 1 (ERAP1) gene (rs27044, rs10050860 and rs30187) and one in the endoplasmic reticulum aminopeptidase 2 (ERAP2) gene (rs2549782), were genotyped and family-based association analyses were performed. RESULTS: Family-based association testing (FBAT -e; empirical variance option) analysis showed that ERAP1 rs30187[T] was associated with AS (additive model: p=0.02; dominant model: p=0.007). Haplotype permutation tests (HBAT-p) showed that a haplotype in the ERAP1 and ERAP2 locus (rs27044[G] rs30187[T] rs2549782[T]) was significantly associated with AS (two-sided p value by permutation test 0.009 for additive and 0.008 for dominant model, respectively). CONCLUSION: This study shows that one ERAP1 SNP and a haplotype in the ERAP1 and ERAP2 locus are associated with familial AS.

The CPPDD-associated ANKH M48T mutation interrupts the interaction of ANKH with the sodium/phosphate cotransporter PiT-1.

OBJECTIVE: Numerous dominant human homolog of progressive ankylosis (ANKH) mutations have been identified in familial calcium pyrophosphate dihydrate crystal deposition disease (CPPDD). Due to the dominant nature of these mutations, we investigated whether ANKH interacts with other proteins; and if so, whether any CPPDD-associated ANKH mutation might disrupt such protein interactions. METHODS: Stable ATDC5 ANKH wt- and ANKH M48T-transfectants were generated. Lysates from these transfectants were used to identify candidate protein interaction with ANKH by coimmunoprecipitation followed by Western blot analysis. The effect of high phosphate on the expression of genes involved in modulating Pi (inorganic phosphate)/PPi (inorganic pyrophosphate) homeostasis in these transfectants was assessed. RESULTS: We showed that ANKH protein associates with the sodium/phosphate cotransporter PiT-1, and that ANKH M48T mutant protein failed to interact with PiT-1. We also showed that upon high phosphate treatment, the normally coordinated upregulation of endogenous Ank and PiT1 transcript expression was disrupted in ANKH M48T transfectants. CONCLUSION: Our results suggested that there is a coordinated interrelationship between 2 key participants of Pi and PPi metabolism, ANKH and PiT-1.

The ANKH ΔE490Mutation in Calcium Pyrophosphate Dihydrate Crystal Deposition Disease (CPPDD) affects tissue non-specific Alkaline Phosphatase (TNAP) activities.

ANKH (human homolog of progressive ankylosis) regulates inorganic pyrophosphate (PPi) transport. Dominant ANKH mutations were detected in at least five multiplex families with calcium pyrophosphate dihydrate crystal deposition disease (CPPPD). The objective of this study is to assess the functional consequences of one CPPDD-associated ANKH mutation (ΔE490) in chondrogenic ATDC5 cells. Stable ATDC5 transfectants bearing myc-tagged constructs of wild-type ANKH, mutant ANKH (ΔE490) and neo controls were generated. Upon ITS (insulin, transferrin and selenium) induction, expression of chondrocyte markers including alkaline phosphatase activity in the various transfectants was assessed. The ANKH ΔE490- transfectants had low alkaline phosphatase activities throughout ITS treatment due to lower TNAP protein expression and the presence of intracellular low-molecular-weight inhibitors. Our results suggest that the interplay of ANKH and TNAP activities is tightly regulated.

Toll-like receptor 2 variants are associated with acute reactive arthritis.

OBJECTIVE: We previously reported a recent outbreak of salmonellosis in which some individuals developed complications of the enteric infection. The objective of this study was to identify genetic variants that might predispose infected individuals to develop articular and/or extraarticular sequelae after Salmonella enteritidis infection. METHODS: The entire exposed cohort was invited to participate in the study by sending a saliva sample for DNA analysis. Seventy-five Salmonella-infected subjects for whom there was clinical information agreed to participate and were stratified into 4 groups. Group 1 patients had arthritis and extraarticular features, group 2 patients had arthritis alone, group 3 patients had extraarticular features alone, and group 4 patients had neither. DNA samples from an uninfected cohort of 91 normal subjects were also genotyped. Genotyping was performed using 2 Toll-like receptor 2 (TLR-2) (rs5743708 and rs5743704) and 2 TLR-4 (rs4986790 and rs4986791) single-nucleotide polymorphisms (SNPs). Statistical analyses were carried out using chi-square tests. RESULTS: There was no association of TLR-4 exonic variants with any clinical events that were reported as accompanying the Salmonella infection. In contrast, compared with normal controls, one of the rare TLR-2 SNPs (rs5743708, R753Q) was associated with the development of arthritis and extraarticular features (P = 0.015 by chi-square test). The TLR-2 variant 753Q was not detected in any of the infected individuals with an uncomplicated course. Another TLR-2 variant, 631H, was associated with articular symptoms in infected males (P = 0.03 by chi-square test). CONCLUSION: In this outbreak, genetic variants of TLR-2, but not TLR-4, were associated with acute reactive arthritis following infection with S enteritidis.

Symptomatic acute reactive arthritis after an outbreak of salmonella.

OBJECTIVE: In 2005, 592 individuals in Ontario developed acute gastroenteritis, predominantly after consuming bean sprouts contaminated with Salmonella enteritidis. Salmonella is a known trigger of reactive arthritis (ReA). We describe the population affected by the Salmonella outbreak in terms of clinical presentation of self-reported arthritic symptoms and HLA-B27 genotyping. METHODS: Subjects were mailed a questionnaire, which assessed symptoms consistent with ReA. Subsequently, subjects were asked to submit saliva samples, which were analyzed for HLA-B27. Simple descriptive statistics were performed for analysis of survey responses, and the genetic component was analyzed by chi-square or Fisher's exact tests. RESULTS: Most respondents were female (71.3%), with a mean age of 46.0 years. The mean duration of diarrhea symptoms was 16.5 days. 62.5% of respondents reported extraintestinal symptoms that were consistent with ReA. The most commonly reported features were joint pain, swelling or stiffness (46.2%), stiffness > 30 min (35.6%), ocular symptoms (24.0%), and visibly swollen joints (19.2%). Subjects with Salmonella infection had a similar incidence of HLA-B27, regardless of whether they developed symptoms consistent with ReA or not. Notably, HLA-B27 was present more frequently in those who developed Salmonella infection than in healthy controls (OR 3.0). CONCLUSION: The study, one of the largest for a dysenteric outbreak, revealed a high event rate of self-reported symptoms consistent with ReA in those infected with Salmonella. Our results showed that HLA-B27 may have rendered individuals more susceptible to Salmonella infection, but did not contribute to the development of symptoms consistent with ReA after infection. We note that the methods used in this study, including self-report, are not ideal for diagnosis of inflammatory arthritis. However, given the rarity of large outbreaks of Salmonella, the study adds valuable knowledge about the course of ReA.

Retinol (vitamin A) and retinol-binding protein levels are decreased in ankylosing spondylitis: clinical and genetic analysis.

OBJECTIVE: Retinol (vitamin A) plays an important role in bone structure and function. Treatment with retinoids has been associated with bone abnormalities mimicking spondyloarthropathy and diffuse idiopathic skeletal hyperostosis. To determine whether retinol concentrations are altered in patients with ankylosing spondylitis (AS), we examined serum retinol levels in patients with AS and healthy controls. METHODS: Retinol was assessed using mass spectrometry, and retinol-binding protein levels were assessed by ELISA. Retinol levels were correlated with clinical disease activity indices. The CYP26 gene, which plays a key role in retinol metabolism, was examined to define any single-nucleotide polymorphisms (SNP) associations with AS. RESULTS: Retinol levels were significantly lower in the AS cohort than in controls (mean 2.39 +/- 0.88 micromol/l for AS, 3.34 +/- 1.01 micromol/l for controls; p < 0.0001). Retinol-binding protein levels were also lower in AS than controls (AS 4.65 +/- 2.10 microg/l; controls 7.48 +/- 4.87 microg/l; p < 0.001). Serum retinol levels did not correlate with indices of disease activity defined serologically (C-reactive protein, erythrocyte sedimentation rate) or clinically (Bath AS Disease Activity Index, Bath AS Functional Index). Genetic analysis showed that an exonic CYP26C1 SNP (rs11187265) is not associated with AS. CONCLUSION: The hallmark of AS is neo-ossification. AS is associated with abnormal serum levels of retinol, a biochemical factor linked to pathological hyperostosis. Further genetic studies are warranted into the genetic basis of the retinol-AS relationship.

Microcytosis in ank/ank mice and the role of ANKH in promoting erythroid differentiation.

Progressive ankylosis (Ank and the human homolog, ANKH) is a transmembrane protein which regulates transport of inorganic pyrophosphate (PPi). ank/ank mice with a mutated ank gene, have calcification and bone ankylosis of the affected joints. In the course of studying these mutant mice, we found that they have microcytosis. These mutant mice have lower mean red blood cell volume (MCV) and lower hemoglobin content in red cells (mean corpuscular hemoglobin, MCH) than normal mice. Using quantitative real-time PCR analysis, we showed that Ank was expressed in the E/Meg bipotent precursor, BFU-E, CFU-E, but there was no Ank expression in the hemoglobinizing erythroblasts. Stable ANKH transfectants in K562 cells highly expressed two immature erythroid cell markers, E-cadherin and endoglin. Enhanced Erythropoietin (Epo) expression and downregulation of SHP-1 were detected in these transfectants. Consequently, the autocrine Epo-EpoR signaling pathway was activated, as evidenced by higher p-Tyr JAK2, p-Tyr EpoR and p-Tyr STAT5B in the ANKH transfectants. Our results revealed a novel function of ANKH in the promotion of early erythroid differentiation in K562 cells. We also showed that ank/ank mice have lower serum levels of Epo than the normal littermates, and this is the likely cause of microcytosis in these mutant mice.

Association of a TNAP haplotype with ankylosing spondylitis.

OBJECTIVE: To use a candidate gene approach to the identification of genetic markers that are significantly associated with ankylosing spondylitis (AS). METHODS: We genotyped 201 multiplex AS families with 1 exonic and 5 intronic single-nucleotide polymorphisms (SNPs) in TNAP, the gene that encodes tissue-nonspecific alkaline phosphatase, and performed family-based association analyses. RESULTS: In our cohort of 201 multiplex AS families, the TNAP haplotype rs3767155 (G)/rs3738099 (G)/rs1780329 (T) was significantly associated with AS (P = 0.032 by additive model). Haplotype-Based Association Testing (HBAT) analyses of AS families in which both men and women were affected showed that the same TNAP haplotype was significantly associated with AS (P = 0.002 by additive model). Using setafftrait code 1 0 0 in the HBAT program, testing specifically for affected men in AS families containing affected individuals of both sexes, this TNAP haplotype was also significantly associated with AS (P = 0.001 by additive model). The HBAT -p option (haplotype permutation test) was used to compute the "exact" P value via a Monte Carlo method for each haplotype (haplotype permutation test) and for the minimum observed P value among the haplotypes (whole marker permutation using the minimal P test), and both P values were statistically significant (2-sided P value for haplotype rs3767155 [G]/rs3738099 [G]/rs1780329 [T] = 0.00059, the smallest observed P value among all the individual haplotype scores = 0.003). Interestingly, this haplotype was not associated with AS in affected women from the same families. CONCLUSION: Our results indicate that the TNAP haplotype rs3767155 (G)/rs3738099 (G)/rs1780329 (T) is a novel genetic marker in men that is significantly associated with AS in multiplex families containing affected individuals of both sexes.

Investigations into the regulation and function of the SH2 domain-containing protein-tyrosine phosphatase, SHP-1.

Our laboratory is interested in identifying genes relevant to diseases. Our approach is to use spontaneous mouse mutants with immunological defects and decipher the molecular basis of the phenotypes. In the early 1990s, our attention was focused on the motheaten and viable motheaten mouse mutants. We used these mutant mice as a model system for elucidating the genetic and cellular events contributing to expression of normal hematopoietic and immune function. Our initial goal was to identify the gene responsible for the motheaten and viable motheaten phenotype. In 1993, we and others reported that both motheaten and viable motheaten mice have mutations in the SHP-1 gene. Currently, there are more than 600 publications involving SHP-1. In this review, rather than summarizing all these studies, we highlight work involving SHP-1 that were/are carried out in our and our collaborators' laboratories.

ANKH variants associated with ankylosing spondylitis: gender differences.

The ank (progressive ankylosis) mutant mouse, which has a nonsense mutation in exon 12 of the inorganic pyrophosphate regulator gene (ank), exhibits aberrant joint ankylosis similar to human ankylosing spondylitis (AS). We previously performed family-based association analyses of 124 Caucasian AS families and showed that novel genetic markers in the 5' flanking region of ANKH (the human homolog of the murine ank gene) are modestly associated with AS. The objective of the present study was to conduct a more extensive evaluation of ANKH variants that are significantly associated with AS and to determine whether the association is gender specific. We genotyped 201 multiplex AS families with nine ANKH intragenetic and two flanking microsatellite markers, and performed family-based association analyses. We showed that ANKH variants located in two different regions of the ANKH gene were associated with AS. Results of haplotype analyses indicated that, after Bonferroni correction, the haplotype combination of rs26307 [C] and rs27356 [C] is significantly associated with AS in men (recessive/dominant model; P = 0.004), and the haplotype combination of rs28006 [C] and rs25957 [C] is significantly associated with AS in women (recessive/dominant model; P = 0.004). A test of interaction identified rs26307 (i.e. the region that was associated in men with AS) as showing a difference in the strength of the association by gender. The region associated with AS in women only showed significance in the test of interaction among the subset of families with affected individuals of both genders. These findings support the concept that ANKH plays a role in genetic susceptibility to AS and reveals a gender-genotype specificity in this interaction.

Expression of cytokines and receptors in normal, immortalized, and malignant ovarian epithelial cell lines.

BACKGROUND: Ovarian carcinoma originates from the epithelial cells on the surface of the ovary. This study evaluates cytokine production by these cells. MATERIALS AND METHODS: Normal human ovary surface epithelial cells (HOSE cells), immortalized HOSE cells and ovarian cancer cells were used for the study of cytokines. RESULTS: Eight of 14 cytokines were increased in > or = 3 ovarian cancer cell lines compared with normal HOSE cells. Three cytokines were increased 5-fold in the immortalized HOSE cell line and in multiple ovarian cancer cell lines. Cytokine receptor expression revealed that 7 of 8 ovarian cancer cell lines had > or = 1 autocrine loop. Anti-EGFR antibody failed to inhibit growth of ovarian cancer cells which expressed multiple cytokine receptors. CONCLUSION: Ovarian cancer cells produce more cytokines than normal HOSE cells. Immortalized HOSE cells display a cytokine profile similar to the cancer cells. Finally, multiple autocrine loops in ovarian cancer may limit the therapeutic usefulness of single cytokine receptor blockade.

Novel genetic markers in the 5'-flanking region of ANKH are associated with ankylosing spondylitis.

OBJECTIVE: To use a candidate gene approach for the identification of genetic markers that are significantly linked to and associated with ankylosing spondylitis (AS). METHODS: We searched for novel polymorphisms in the ANKH gene (human homolog of the murine progressive ankylosis gene) and genotyped 2 polymorphic sites, one in the 5'-noncoding region and the other in the promoter region of ANKH, using DNA from affected (n = 273) and unaffected (n = 112) individuals from 124 AS families. We used these ANKH and other nearby polymorphisms to perform linkage and family-based association analyses. RESULTS: We identified 2 novel polymorphic sites: one in the 5'-noncoding region of ANKH involving 1-2 copies of an 8-bp repeat (denoted as ANKH-OR), and the other in the promoter region involving different copy numbers of a triplet repeat (denoted as ANKH-TR). ANKH-OR and ANKH-TR were in complete linkage disequilibrium. Five markers (D5S1953, ANKH-TR, ANKH-OR, D5S1954, and D5S1963) were used for both the linkage and association analyses. Multipoint linkage analysis of 124 AS families showed a modest level of significance (nonparametric linkage score 2.15; P = 0.015) at the ANKH region. The contribution of ANKH to AS susceptibility (lambda(s)) was 1.9. A family-based association study on the same AS families revealed that both ANKH-OR allele 1 and ANKH-TR allele 7 were significantly associated with disease, assuming an additive model (for ANKH-OR allele 1, P = 0.03; for ANKH-TR allele 7, P = 0.04). CONCLUSION: Our results indicate that ANKH-OR and ANKH-TR are novel genetic markers that are significantly associated with AS.