Measuring vitamin D levels: surrogates are insufficient.

AIMS: With the increasing evidence of adverse consequences because of low vitamin D levels on health demand for vitamin D, screening is increasing. The objective of the study was to assess whether parathyroid hormone (PTH) levels/bone profile is sufficient to identify patients with vitamin D insufficiency or deficiency, or whether vitamin D should be measured directly. METHODOLOGY: A total of 1560 serum specimens, with requests for 25-hydroxyvitamin D (25-OH vitamin D), calcium, phosphate, alkaline phosphatase (ALP), creatinine and PTH on the same sample were analysed at Salford Royal Hospital from November 2010 to November 2012. RESULTS: The prevalence of total vitamin D insufficiency or deficiency (defined as total 25-OH vitamin D < 50 nmol/l) was 62.9% (981/1560) overall, with males having higher proportions (67.2 vs. 59.3 per cent; χ(2) = 8.78, p = 0.003). There was no overall trend in mean serum adjusted calcium across categories of 25-OH vitamin D status but mean serum phosphate was significantly lower (F = 6.53, p < 0.0001) in patients with a 25-OH vitamin D level < 50 nmol/l. However in patients with vitamin D deficiency, a significant proportion had PTH, calcium, phosphate and alkaline phosphatase levels within the laboratory normal range. Even at a 25-OH vitamin D < 10 nmol/l, 71.6% had a normal PTH, 89.8% had normal serum calcium levels, 84.9% had normal phosphate levels and 81.6% had normal serum ALP. CONCLUSIONS: Therefore, despite the costs associated with the measurement of vitamin D, our findings show that no surrogate is adequate for screening for vitamin D deficiency.

Network analysis: a new approach to study endocrine disorders.

Systems biology is the study of the interactions that occur between the components of individual cells - including genes, proteins, transcription factors, small molecules, and metabolites, and their relationships to complex physiological and pathological processes. The application of systems biology to medicine promises rapid advances in both our understanding of disease and the development of novel treatment options. Network biology has emerged as the primary tool for studying systems biology as it utilises the mathematical analysis of the relationships between connected objects in a biological system and allows the integration of varied 'omic' datasets (including genomics, metabolomics, proteomics, etc.). Analysis of network biology generates interactome models to infer and assess function; to understand mechanisms, and to prioritise candidates for further investigation. This review provides an overview of network methods used to support this research and an insight into current applications of network analysis applied to endocrinology. A wide spectrum of endocrine disorders are included ranging from congenital hyperinsulinism in infancy, through childhood developmental and growth disorders, to the development of metabolic diseases in early and late adulthood, such as obesity and obesity-related pathologies. In addition to providing a deeper understanding of diseases processes, network biology is also central to the development of personalised treatment strategies which will integrate pharmacogenomics with systems biology of the individual.

Functional evaluation of TNFAIP3 (A20) in rheumatoid arthritis.

OBJECTIVES: To determine the protein expression of TNFAIP3 in synovium and to show the capability of 6q23 intergenic SNPs, associated with rheumatoid arthritis (RA) susceptibility, to influence TNFAIP3 gene transcription. METHODS: Immunohistochemistry for TNFAIP3, NF-kB p65 and phosphorylated NF-kB p65 protein expression was performed in 6 RA knee joint synovium samples compared to 9 osteoarthritis (OA) samples. Luciferase reporter gene assays were used to examine the regulatory ability of RA associated SNP variants on TNFAIP3 promoter activity. Sense and antisense constructs were prepared for rs6920220 alleles, together with each of the 4 SNPs in r2=1 with it (rs6933404, rs2327832, rs6927172 and rs17264332), coupled to the TNFAIP3 promoter. Transient transfections were performed in a human T lymphoblastoid (CEMC7A) cell line. Bioinformatic software was utilised to prioritise SNPs for further investigation. Electrophoretic mobility shift assays (EMSA), using CEMC7A nuclear extracts, were conducted for the rs6927172 SNP alleles. RESULTS: TNFAIP3 protein expression was seen in the synovium samples and differential TNFAIP3 protein expression between RA vs. OA synoviocytes observed. Within RA synoviocytes TNFAIP3 expression is predominately cytoplasmic, whereas in OA its expression is strongly nuclear and cytoplasmic. For 3 of the 5 SNPs investigated (rs6920220, rs6933404, rs6927172) evidence of repressor activity of TNFAIP3 transcription was seen and EMSA data showed evidence of differential transcription factor binding to rs6927172 alleles. CONCLUSIONS: This is the first observation of TNFAIP3 protein expression in RA and OA synovium. In vitro analysis of 6q23 intergenic SNPs supports the possibility of the functional regulation of TNFAIP3.

Interferon-inducible factor 16 is a novel modulator of glucocorticoid action.

Previously, we used cDNA expression profiling to identify genes associated with glucocorticoid (Gc) sensitivity. We now identify which of these directly influence Gc action. Interferon-inducible protein 16 (IFI16), bone morphogenetic protein receptor type II (BMPRII), and regulator of G-protein signaling 14 (RGS14) increased Gc transactivation, whereas sialyltransferase 4B (SIAT4B) had a negative effect. Amyloid beta (A4) precursor-protein binding, family B, member 1 (APBB1/Fe65) and neural cell expressed developmentally down-regulated 9 (NEDD9) were without effect. Only IFI16 potentiated Gc repression of NF-kappaB. In addition, IFI16 affected basal expression, and Gc induction of endogenous target genes. IFI16 did not affect glucocorticoid receptor (GR) expression, ligand-dependent repression of GR expression, or the ligand-dependent induction of GR phosphorylation on Ser-211 or Ser-203. Coimmunoprecipitation revealed an interaction, suggesting that IFI16 modulation of GR function is mediated by protein crosstalk. Transfection analysis with GR mutants showed that the ligand-binding domain of GR binds IFI16 and is the target domain for IFI16 regulation. Analysis of human lung sections identified colocalization of GR and IFI16, suggesting a physiologically relevant interaction. We demonstrate that IFI16 is a novel modulator of GR function and show the importance of analyzing variation in Gc sensitivity in humans, using appropriate technology, to drive discovery.

Autoinflammatory genes and susceptibility to psoriatic juvenile idiopathic arthritis.

OBJECTIVE: To investigate the association of NLRP3, NOD2, MEFV, and PSTPIP1, genes that cause 4 of the autoinflammatory hereditary periodic fever syndromes (HPFS), with juvenile idiopathic arthritis (JIA). METHODS: Fifty-one single-nucleotide polymorphisms (SNPs) across the 4 loci were investigated using MassArray genotyping in 950 Caucasian patients with JIA living in the UK and 728 ethnically matched healthy controls. RESULTS: Prior to Bonferroni correction for multiple testing, significant genotype associations between 6 SNPs in MEFV and JIA were observed and, in subgroup analysis, associations between 12 SNPs across all 4 loci and the subgroup of patients with psoriatic JIA were found. After Bonferroni correction for multiple testing, 2 genotype associations remained significant in the subgroup of patients with psoriatic JIA (MEFV SNP rs224204 [corrected P = 0.025] and NLRP3 SNP rs3806265 [corrected P = 0.04]). CONCLUSION: These findings support the use of monogenic loci as candidates for investigating the genetic component of complex disease and provide preliminary evidence of association between SNPs in autoinflammatory genes and psoriatic JIA. Our findings raise the interesting possibility of a shared disease mechanism between the HPFS and psoriatic JIA, potentially involving abnormal production of interleukin-1beta.

Glucocorticoids suppress macrophage migration inhibitory factor (MIF) expression in a cell-type-specific manner.

MIF is a potent proinflammatory cytokine involved in inflammatory arthritis. Glucocorticoids (GC) have been reported to induce secretion of MIF in rodent cells, and as MIF counteracts the anti-inflammatory effects of GC, this has implications for human inflammatory disease. Transient transfection studies showed that the MIF promoter was repressed by dexamethasone (Dex) (10 nM) in CEM C7A cells, with up to 50% suppression by 100 nM. However, there was no regulation of the promoter by GC in A549 cells. We also found that subnanomolar concentrations of Dex suppressed MIF secretion, measured by ELISA, by 80% in both human T lymphoblasts (CEM C7A) and human lung epithelial cells (A549). Endogenous MIF mRNA was also repressed by GC in CEM C7A cells, measured both by Northern blot and quantitative RT-PCR assays, but there was no such regulation in A549 cells. This suggests that GC affects translation rather than transcription of MIF in A549 cells. These results contradict earlier results with the rat cell line RAW 264.7. Therefore, we analysed MIF secretion from RAW 264.7 cells but found no GC effect on secretion. Understanding how GC regulates MIF in a cell-type-dependent manner may give insights into GC-refractory human inflammatory diseases.

Macrophage migration inhibitory factor: molecular, cellular and genetic aspects of a key neuroendocrine molecule.

The immunological and neuroendocrine properties of macrophage migration inhibitory factor (MIF) are diverse. In this article we review the known cellular, molecular and genetic properties of MIF that place it as a key regulatory cytokine, acting within both the innate and adaptive immune responses. The unexpected and paradoxical induction of MIF secretion by low concentrations of glucocorticoids is explored. The role of MIF as a locally acting modulator of glucocorticoid sensitivity within foci of inflammation is also discussed. MIF has no homology with any other pro-inflammatory cytokine and until recently lacked a recognised transmembrane receptor. MIF has also been shown to be directly taken up into target cells and to interact with intracellular signalling molecules, including the Jun activation domain-binding protein Jab-1.Comprehensive analysis of the MIF gene has identified important functional polymorphisms and a series of genetic studies has revealed both association and linkage of MIF with inflammatory diseases. Altered MIF regulation may therefore be pivotal to acquiring chronic inflammation following an innate immune response.

The role of the PTPRC (CD45) mutation in the development of multiple sclerosis in the North West region of the United Kingdom.

BACKGROUND: A point mutation in protein tyrosine phosphatase receptor, type c polypeptide (PTPRC) has been associated with familial multiple sclerosis. This CG mutation at position 77 of exon 4 results in altered expression of CD45 isoforms on immune cells. OBJECTIVE: To study the incidence of PTPRC mutations in subjects with multiple sclerosis in the North West region of the United Kingdom. METHODS: Affected and unaffected subjects from five pedigrees with familial multiple sclerosis, 330 non-familial cases of multiple sclerosis, and 197 controls were studied. Genomic DNA was amplified using CD45IE34 and CD45IE44 primers, digested with Mspl, and run on an agarose gel. Polymerase chain reaction products were sequenced to exclude any other mutations. RESULTS: No PTPRC exon 4 genomic mutations were seen in any of the five families. In the non-familial cases the incidence of mutation was 4.1% in 197 controls and 5.1% in 330 multiple sclerosis patients. No significant association was found in this study with this mutation and disease susceptibility, sex, or an extended disability scale score of < 5.5. CONCLUSIONS: This candidate does not appear to influence the development of familial multiple sclerosis in this population. The negative result could arise from a type II error owing to the number of families and non-familial cases screened. Alternatively it might suggest that the contribution of the PTPRC mutation depends upon the genetic background.

Neuroendocrine gene polymorphisms and susceptibility to juvenile idiopathic arthritis.

OBJECTIVE: To investigate the involvement of neuroendocrine candidate genes in the aetiopathogenesis of juvenile idiopathic arthritis (JIA). METHODS: Single-nucleotide polymorphisms and intragenic microsatellite markers within five neuroendocrine candidate genes (CRH, CBG, CYP19, ESR1, PRL) were investigated in 463 clinically characterized UK Caucasian JIA patients and a panel of 276 unrelated, healthy UK Caucasian controls. RESULTS: None of the polymorphisms investigated showed any statistically significant associations with JIA. CONCLUSIONS: The lack of association with polymorphisms of these neuroendocrine genes suggests that they are not involved in susceptibility to JIA.

A novel 5'-flanking region polymorphism of macrophage migration inhibitory factor is associated with systemic-onset juvenile idiopathic arthritis.

OBJECTIVE: To determine if polymorphisms of the macrophage migration inhibitory factor (MIF) gene are associated with systemic-onset juvenile idiopathic arthritis (JIA). METHODS: Denaturing high-performance liquid chromatography was used to screen for the MIF gene in 32 healthy Caucasian subjects. One hundred seventeen UK Caucasian patients with systemic-onset JIA and 172 unrelated healthy UK Caucasian controls were genotyped for a single-nucleotide polymorphism (SNP) identified in the 5'-flanking region of the gene, using polymerase chain reaction-restriction fragment length analysis. RESULTS: A G-to-C transition was identified at position -173 of the MIF gene. The presence of a C at -173 creates an activator protein 4 transcription factor binding site. Allele and genotype frequencies differed significantly between the patients and controls for the MIF-173 polymorphism. Individuals possessing a MIF-173*C allele have an increased risk of systemic-onset JIA (36.8% versus 20.3%) (odds ratio 2.3, 95% confidence interval 1.34-3.86; P = 0.0005). CONCLUSION: This is the first report of a SNP in the MIF gene. This polymorphism is associated with systemic-onset JIA.

Cytokine gene polymorphisms and susceptibility to juvenile idiopathic arthritis. British Paediatric Rheumatology Study Group.

OBJECTIVE: To investigate the involvement of candidate cytokine genes in the pathogenesis of juvenile idiopathic arthritis (JIA). METHODS: Single nucleotide polymorphisms and intragenic microsatellite markers within 8 candidate cytokine genes (interleukin-1alpha [IL-1alpha], IL-2, IL-4, IL-6, IL-10, interferon-alpha1 [IFNA1], interferon-gamma [IFNG], and interferon regulatory factor 1 [IRF-1]) were investigated in 417 Caucasian patients with clinically characterized JIA and a panel of 276 unrelated, healthy Caucasian controls, all from the United Kingdom. RESULTS: A novel 3'-untranslated region (3'UTR) polymorphism in IRF-1 was found to be associated with susceptibility to JIA (corrected P = 0.002). No significant association with IL-1alpha, IL-2, IL-4, IL-6, IL-10, IFNA1, or IFNG was observed. CONCLUSION: An association between JIA and a previously unreported 3'UTR polymorphism of IRF-1 was observed. This association was not found to be specific to any particular JIA subgroup. This suggests that IRF-1 may contribute to a common pathogenesis shared by all JIA patients, regardless of clinical phenotype. This is most likely to be a genetic contribution to the chronic inflammatory process that underlies JIA pathology.

Subtyping of juvenile idiopathic arthritis using latent class analysis. British Paediatric Rheumatology Group.

OBJECTIVE: To use statistical techniques to identify underlying subtypes of juvenile idiopathic arthritis (JIA) that best explain the observed relationships of clinical and laboratory variables, and to compare the statistically derived subtypes with those defined by the International League of Associations for Rheumatology (ILAR) criteria and examine them for HLA associations. METHODS: Information on 572 patients diagnosed as having JIA was summarized by 10 clinical and laboratory categorical variables (age at onset, large joint involvement, small joint involvement, polyarthritis, symmetric arthritis, spinal pain, fever, psoriasis, antinuclear antibodies [ANA], and rheumatoid factor). Latent class analysis (LCA) was used to identify underlying ("latent") classes that explained the relationships among the observed variables. Statistical models incorporating 5-8 latent classes were applied to the data. RESULTS: The 7-class model was the most appropriate. Patterns of joint involvement and the presence of ANA were influential in determining latent classes. There was some correspondence between the latent classes and the ILAR categories, but they did not coincide completely. Significant differences between the latent classes were seen for 3 HLA haplotypes (DRB1*04-DQA1*03-DQB1*03, DRB1*13-DQA1*01-DQB1*06, and DRB1*08-DQA1*0401-DQB1*0402). CONCLUSION: LCA provides a novel approach to the task of identifying homogeneous subtypes within the umbrella of JIA. In further work, the identified latent classes will be examined for associations with other candidate genes and for differences in outcome.

Interleukin-10 (IL10) promoter polymorphisms and multiple sclerosis.

Interleukin-10 (IL10) is an anti-inflammatory cytokine which may modulate disease expression in multiple sclerosis (MS). Three dimorphic polymorphisms within the IL10 promoter region at positions - 1082, -819 and -519 have previously been identified. The - 1082*A allele has been associated with low and the - 1082*G allele with high in vitro IL10 production. We have genotyped 185 Caucasian MS patients and 211 ethnically matched controls for each of these three dimorphisms. MS patients were stratified for severity of disease outcome. No associations were found for any IL10 promoter polymorphisms when the MS cases were compared with controls or with disease outcome with regards to disability. IL10 polymorphism does not appear to be associated with MS or to influence disease progression.

Absence of association between interleukin 1 alpha and oligoarticular juvenile chronic arthritis in UK patients.

OBJECTIVE: To determine whether interleukin 1 alpha (IL-1alpha) polymorphisms are associated with UK oligoarticular juvenile chronic arthritis (JCA). PATIENTS AND CONTROLS: A well-characterized population of 164 UK Caucasian oligo-JCA patients and a control panel of 173 unrelated healthy UK Caucasian individuals. METHODS: The IL-1alpha promoter mutation at -889 was examined using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. The cases and controls were also genotyped for an IL-1alpha intronic microsatellite repeat. RESULTS: No association was observed between IL-1alpha polymorphisms and UK oligoarticular JCA patients. In particular, no association between IL-1alpha polymorphisms and chronic anterior uveitis was found. CONCLUSIONS: IL-1alpha is not associated with oligoarticular JCA in UK patients. This differs markedly to findings for IL-1alpha in Norwegian JCA patients.

Absence of an association between mannose-binding lectin polymorphism and rheumatoid arthritis.

It has been proposed that mannose-binding lectin (MBL) interactions with agalactosyl forms of IgG immunoglobulins found in rheumatoid synovial fluid might lead to enhanced complement activation, an important mediator of the joint damage in rheumatoid arthritis (RA). In order to investigate this possible link between increased MBL-mediated activation of complement and perpetuation of rheumatoid synovitis, we have compared the frequency of an allelic form of MBL, known to be incapable of activating complement, in a group of hospital patients with severe RA and control subjects. No evidence was found to support an association between the presence of this MBL allele and protection from rheumatoid disease; genotype frequencies were similar in both groups. This suggests that complement activation via MBL-agalactosyl IgG complexes is unlikely to play a major role in the pathophysiology of RA.

TAP2D is associated with HLA-B44 and DR4 and may contribute to rheumatoid arthritis and Felty's syndrome susceptibility.

OBJECTIVES: TAP2 transporter gene polymorphisms have been ascertained in patients with rheumatoid arthritis (RA) and Felty's syndrome (FS) to determine whether particular alleles of this gene are disease associated. METHODS: TAP2 dimorphisms at amino acid positions 379, 565 and 665 were detected using ARMS-PCR in 89 RA patients, 24 FS patients and 64 control subjects. TAP 2 alleles were assigned from these results. RESULTS: The frequency of one particular allele, TAP2D, was increased in both RA (OR 2.6, 95% CI 1.2 - 5.8) and FS (OR 3.9, 95% CI 1.4 - 10.7). When individual amino acid polymorphisms were compared between patients and controls, isoleucine at position 379 (present in TAP2D and TAP2C) was significantly increased, indicating that this dimorphism itself may be associated with RA (OR 5.0, 95% CI 2.4 - 10.2) and FS (OR 5.0, 95% CI 1.91 - 3.2). DISCUSSION: The presence of TAP2D was greatly increased in HLA-B44/DR4 positive RA (83%) and FS (67%) patients. These frequencies were appreciably higher than in the HLA-B44/DR4 controls (11%), suggesting that linkage disequilibrium alone may not explain the increase in TAP2D frequency in patients and that this allele may represent an additional risk factor in these conditions.

Antinuclear antibodies in early onset pauciarticular juvenile chronic arthritis (JCA) are associated with HLA-DQB1*0603: a possible JCA-associated human leucocyte antigen haplotype.

A panel of 43 early onset pauciarticular (EOPA) juvenile chronic arthritis (JCA) patients have been typed for human leucocyte antigens (HLA) DRB1, DPB1, DQA1 alleles, and DQB1*0603 status using molecular-based methods. Increased frequencies of DRB1*08 [odds ratio (OR) 7.7, 95% confidence interval (CI) 2.6-22.3], DRB1*11 (OR 3.1, 95% CI 1.2-8.1), DRB1*1301 (OR 7.7, 95% CI 2.6-22.3), DPB1*0201 (OR 3.5, 95% CI 1.6-8.0), DQA1*0103 (OR 4.4, 95% CI 1.5-13.3), DQA1*0501 (OR 2.9, 95% CI 1.3-6.6), DQA1*0601 (OR 30, 95% CI 3.6-241) and DQB1*0603 (OR 7.3, 95% CI 3.0-17.6) were found in the EOPA-JCA group compared with Caucasoid controls. Stratification of the EOPA-JCA group into antinuclear antibody (ANA) positive (n = 18) and ANA negative (n = 25) individuals revealed that ANA positivity was only associated with DRB1*1301 (OR 4.2, 95% CI 1.0-17.3), DPB1*0201 (OR 4.0, 95% CI 1.0-15.7) and DQB1*0603 (OR 11.5, 95% CI 2.5-53.4). Further analysis of the relative contributions of HLA antigens to ANA status revealed that DQB1*0603 determined the primary HLA effect. No apparent interaction between DQB1*0603 and DRB1*1301 or between DQB1*0603 and DPB1*0201 was found to contribute to the association with ANA. We suggest that those ANA positive individuals with a restricted HLA background, (DQB1*0603 positive), defines a group of EOPA-JCA patients which will be especially valuable in the characterization of the ANA associated with EOPA-JCA.

Absence of TAP 2D in Yoruba Nigerians.

We have characterized TAP allele frequencies in a panel of 71 Yoruba Nigerians using ARMS-PCR. With the exception that TAP 2D was absent in Nigerians, TAP 2 allele frequencies in this population were found to be similar to those in a UK white population. HLA-DR4 also was found to be at a low frequency in Yoruba Nigerians (1.4%). This may reflect the absence of TAP 2D in Nigerians as DR4 and TAP 2D are in linkage disequilibrium in UK Caucasoids. The most frequent TAP 1 allele in Yoruba Nigerians was TAP 1A (49%). However, this value will be an underestimate as TAP1 alleles could not be unequivocally assigned in 41% of subjects using the ARMS-PCR methodology.

Tap gene associations in UK caucasoids.

The authors determined the allele frequencies of the TAP1 and TAP2 transporter genes in a healthy UK Caucasoid population by ARMS-PCR. TAP1A was the most frequent TAP1 allele by far, being present in 76% of subjects. TAP1 alleles could not be assigned in 24% of subjects, since the combinations TAP1A/1b and TAP1C/1D cannot be separated. TAP2A was the most frequent TAP2 alleles (75% of subjects) followed by TAP2B (43%), TAP2C (11%), TAP2D (8%) and TAP2E (6%). The authors also identified an individual with a previously undescribed TAP2 allele, TAP2H (isoleucine at amino acid [aa] 379, alanine at aa 565, alanine at aa 665). It was not possible to assign unequivocally TAP2 alleles in 15 individuals (9%) as TAP2A/D and TAP2C/E cannot be distinguished from each other. To address this problem a separate study of families of rheumatoid arthritis (RA) patients selected for this ambiguity were studied. In all five informative families, TAP2A/2D was confirmed as the combination present. In the population studied no evidence was found for linkage disequilibrium between TAP1 and TAP2 or between the TAP genes and HLA-DP. There was no evidence for extensive linkage disequilibrium between the TAP genes and HLA-DQR, although TAP2B was associated with DRI (delta = 0.056, corrected P < 0.01) and TAP2D with DR4 (delta = 0.018). In the RA families studied, TAP2D was found on DRB1*0401-bearing haplotypes.