Profiling microRNAs in individuals at risk of progression to rheumatoid arthritis.

BACKGROUND: Individuals at risk of rheumatoid arthritis (RA) demonstrate systemic autoimmunity in the form of anti-citrullinated peptide antibodies (ACPA). MicroRNAs (miRNAs) are implicated in established RA. This study aimed to (1) compare miRNA expression between healthy individuals and those at risk of and those that develop RA, (2) evaluate the change in expression of miRNA from "at-risk" to early RA and (3) explore whether these miRNAs could inform a signature predictive of progression from "at-risk" to RA. METHODS: We performed global profiling of 754 miRNAs per patient on a matched serum sample cohort of 12 anti-cyclic citrullinated peptide (CCP) + "at-risk" individuals that progressed to RA. Each individual had a serum sample from baseline and at time of detection of synovitis, forming the matched element. Healthy controls were also studied. miRNAs with a fold difference/fold change of four in expression level met our primary criterion for selection as candidate miRNAs. Validation of the miRNAs of interest was conducted using custom miRNA array cards on matched samples (baseline and follow up) in 24 CCP+ individuals; 12 RA progressors and 12 RA non-progressors. RESULTS: We report on the first study to use matched serum samples and a comprehensive miRNA array approach to identify in particular, three miRNAs (miR-22, miR-486-3p, and miR-382) associated with progression from systemic autoimmunity to RA inflammation. MiR-22 demonstrated significant fold difference between progressors and non-progressors indicating a potential biomarker role for at-risk individuals. CONCLUSIONS: This first study using a cohort with matched serum samples provides important mechanistic insights in the transition from systemic autoimmunity to inflammatory disease for future investigation, and with further evaluation, might also serve as a predictive biomarker.

Differential effects of infliximab on absolute circulating blood leucocyte counts of innate immune cells in early and late rheumatoid arthritis patients.

Anti-tumour necrosis factor (TNF) biologics have revolutionized therapy of rheumatoid arthritis (RA). We compared the effects of infliximab on numbers of circulating leucocyte subsets in early RA (disease/symptom duration of ≤1 year) and late RA patients (>1 year). A control group consisted of early RA patients treated with a combination of methotrexate (MTX) and methylprednisolone. Blood samples were obtained at baseline (pre-therapy) from all RA patients, divided into three groups: (i) late RA receiving infliximab/MTX, (ii) early RA-infliximab/MTX, (iii) early RA-steroid/MTX, and also from follow-up patients at 2 and 14 weeks. Significant differences in absolute counts of monocytes and granulocytes were observed between healthy controls and RA patients. At baseline CD14(bright) monocytes and CD16(+) granulocytes were increased in both early RA and late RA patients. CD4(+) T cells, CD8(+) T cells and B cells were all increased at baseline in early RA, but not in late RA. At 2 weeks following infliximab treatment decreased granulocytes were observed in both early and late RA and decreased natural killer (NK) cells in late RA. CD16(+) granulocytes and NK cells were also decreased at 14 weeks post-infliximab in early RA. Biotinylated infliximab was used to detect membrane-associated TNF (mTNF)-expressing leucocytes in RA patients. CD16(+) granulocytes, NK cells and CD14(dim) monocytes all expressed higher levels of mTNF in RA patients. In summary infliximab is associated with decreased CD16(+) granulocyte and NK cell counts, possibly through binding of mTNF. Differential effects of infliximab between early and late RA suggest that pathogenic mechanisms change as disease progresses.

A novel TNFRSF1A splice mutation associated with increased nuclear factor kappaB (NF-kappaB) transcription factor activation in patients with tumour necrosis factor receptor associated periodic syndrome (TRAPS).

OBJECTIVE: To characterise and investigate the functional consequences of a novel TNFRSF1A splice site mutation causing tumour necrosis factor receptor associated periodic syndrome (TRAPS) in a 16-year-old male patient and his mother. METHODS: Mutational DNA screening was performed in the patient and his mother. Western blotting was used to analyse protein expression levels of TNFR1. A multiplex bead immunoassay was used to quantify serum levels of range of cytokines, and an ELISA-based transcription factor assay to measure nuclear factor (NF)-kappaB transactivation. Serum levels of soluble TNFR1 (sTNFR1) were measured by ELISA and fluorescence-activated cell sorting (FACS) analysis used to measure monocyte TNFR1 cell surface expression. RESULTS: A novel mutation, c.472+1G>A (C158delinsYERSSPEAKPSPHPRG), involving a splice site in intron 4 of TNFRSF1A, was found in the proband and affected mother leading to a 45 nucleotide insertion of intronic DNA into the mRNA, resulting in an in-frame insertion of 15 amino acids in the mature TNFR1 protein and a deletion of a cysteine residue C129 (158) in cysteine rich domain (CRD)3. The patients had reduced serum sTNFR1 and surface expression levels of TNFR1, with marked increases in pro- and anti-inflammatory cytokine. Their peripheral blood mononuclear cells (PBMC) had increased basal NF-kappaB activation compared with healthy controls and also had increased p50 nuclear expression following tumour necrosis factor (TNF) stimulation compared with PBMC from healthy controls, as well as T50M (T79M) and C88R (C117R) patients with TRAPS and patients with rheumatoid arthritis (RA). CONCLUSION: A novel, TRAPS causing, TNFRSF1A splice site mutation is associated with decreased sTNFR1 levels, cell surface and whole cell extract expression and increased NF-kappaB transcription factor activation.

Hearing improvement in a patient with variant Muckle-Wells syndrome in response to interleukin 1 receptor antagonism.

BACKGROUND: Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome, and neonatal onset multisystem inflammatory disease, also called chronic, infantile, neurological, cutaneous, and articular syndrome, are three hereditary autoinflammatory syndromes caused by mutations affecting the CIAS1/NALP3 gene on chromosome 1q44. The proinflammatory cytokine, interleukin 1beta, is believed to have a fundamental role in their pathogenesis. CASE REPORT: The case is described of a 59 year old white woman who presented with increasingly severe MWS-type features over a 15 year period. The response to interleukin 1beta inhibition with anakinra was dramatic, including a reduction in intracranial pressure with associated auditory improvement, as demonstrated by serial audiometry. CONCLUSIONS: The confirmed improvement in hearing after initiation of interleukin 1 receptor antagonism corroborates previous reports that specific blockade of this single cytokine reverses most of the symptoms of this group of CIAS1/NALP3 related autoinflammatory conditions, including the sensorineural deafness, which has not been previously reported.

TNF and TNFR polymorphisms in severe sepsis and septic shock: a prospective multicentre study.

Tumour necrosis factor (TNF) is an important pro-inflammatory cytokine produced in sepsis. Studies examining the association of individual TNF single nucleotide polymorphisms with sepsis have produced conflicting results. This study investigated whether common polymorphisms of the TNF locus and the two receptor genes, TNFRSF1A and TNFRSF1B, influence circulating levels of encoded proteins, and whether individual polymorphisms or extended haplotypes of these genes are associated with susceptibility, severity of illness or outcome in adult patients with severe sepsis or septic shock. A total of 213 Caucasian patients were recruited from eight intensive care units (ICU) in the UK and Australia. Plasma levels of TNF (P = 0.02), sTNFRSF1A (P = 0.005) and sTNFRSF1B (P = 0.01) were significantly higher in those who died on ICU compared to those who survived. There was a positive correlation between increasing soluble receptor levels and organ dysfunction (increasing SOFA score) (sTNFRSF1A R = 0.51, P < 0.001; sTNFRSF1B R = 0.53, P < 0.001), and in particular with the degree of renal dysfunction. In this study, there were no significant associations between the selected candidate TNF or TNF receptor polymorphisms, or their haplotypes, and susceptibility to sepsis, illness severity or outcome. The influence of polymorphisms of the TNF locus on susceptibility to, and outcome from sepsis remains uncertain.

The EIF2AK3 gene region and type I diabetes in subjects from South India.

Mutations in the EIF2AK3 gene underlie susceptibility to the Wolcott-Rallison syndrome, which is a monogenic disease associated with insulin-deficient neonatal diabetes. Furthermore, suggestive evidence of linkage between type 1 diabetes (T1DM) and the EIF2KA3 chromosomal region has been reported in Scandinavian families. We have investigated the hypothesis that polymorphic variants in and around the EIF2AK3 gene might partially account for susceptibility to T1DM in South Indian subjects. Excess transmission of the common alleles of two polymorphic markers (D2S1786 and 15INDEL, located within the gene) downstream of EIF2AK3, either singly (D2S1786, P = 0.01) and 15INDEL (P = 0.02) or as a combination (P < 0.001), were found in 234 families with a T1DM proband. There was also a clear paternal effect for the 15INDEL marker (P = 0.005) on disease susceptibility. The presence of the common allele of both markers was found in decreased frequency in the subjects with normal glucose tolerance compared to probands with T1DM (both P

Allelic variants in genes associated with hereditary periodic fever syndromes as susceptibility factors for reactive systemic AA amyloidosis.

We investigated the hypothesis that low-penetrance mutations in genes (TNFRSF1A, MEFV and NALP3/CIAS1) associated with hereditary periodic fever syndromes (HPFs) might be risk factors for AA amyloidosis among patients with chronic inflammatory disorders, including rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), Crohn's disease, undiagnosed recurrent fevers and HPFs themselves. Four of 67 patients with RA plus amyloidosis had MEFV variants compared with none of 34 RA patients without amyloid (P value=0.03). The E148Q variant of MEFV was present in two of the three patients with TNF receptor-associated periodic syndrome (TRAPS) complicated by amyloid in two separate multiplex TRAPS families containing 5 and 16 affected members respectively, and the single patient with Muckle-Wells syndrome who had amyloidosis was homozygous for this variant. The R92Q variant of TNFRSF1A was present in two of 61 JIA patients with amyloidosis, and none of 31 nonamyloidotic JIA patients. No HPF gene mutations were found in 130 healthy control subjects. Although allelic variants in HPFs genes are not major susceptibility factors for AA amyloidosis in chronic inflammatory disease, low-penetrance variants of MEFV and TNFRSF1A may have clinically significant proinflammatory effects.

Hyper IgD syndrome (HIDS) associated with in vitro evidence of defective monocyte TNFRSF1A shedding and partial response to TNF receptor blockade with etanercept.

Hereditary periodic fever syndromes comprise a group of distinct disease entities linked by the defining feature of recurrent febrile episodes. Hyper IgD with periodic fever syndrome (HIDS) is caused by mutations in the mevalonate kinase (MVK) gene. The mechanisms by which defects in the MVK gene cause febrile episodes are unclear and there is no uniformly effective treatment. Mutations of the TNFRSF1A gene may also cause periodic fever syndrome (TRAPS). Treatment with the TNFR-Fc fusion protein, etanercept, is effective in some patients with TRAPS, but its clinical usefulness in HIDS has not been reported. We describe a 3-year-old boy in whom genetic screening revealed a rare combination of two MVK mutations producing clinical HIDS as well as a TNFRSF1A P46L variant present in about 1% of the population. In vitro functional assays demonstrated reduced receptor shedding in proband's monocytes. The proband therefore appears to have a novel clinical entity combining Hyper IgD syndrome with defective TNFRSF1A homeostasis, which is partially responsive to etanercept.

Genetics of type 1 diabetes mellitus.

At least 20 different chromosomal regions have been linked to type 1 diabetes (T1D) susceptibility in humans, using genome screening, candidate gene testing, and studies of human homologues of mouse susceptibility genes. The largest contribution from a single locus (IDDM1) comes from several genes located in the MHC complex on chromosome 6p21.3, accounting for at least 40% of the familial aggregation of this disease. Approximately 30% of T1D patients are heterozygous for HLA-DQA1*0501-DQB1*0201/DQA1*0301-DQB1*0302 alleles (formerly referred to as HLA-DR3/4 and for simplification usually shortened to HLA-DQ2/DQ8), and a particular HLA-DQ6 molecule (HLA-DQA1*0102-DQB1*0602) is associated with dominant protection from the disease. There is evidence that certain residues important for structure and function of both HLA-DQ and DR peptide-binding pockets determine disease susceptibility and resistance. Independent confirmation of the IDDM2 locus on chromosome 11p15.5 has been achieved in both case-control and family-based studies, whereas associations with the other potential IDDM loci have not always been replicated. Several possibilities to explain these variable results from different studies are discussed, and a key factor affecting both linkage and association studies is that the genetic basis of T1D susceptibility may differ between ethnic groups. Some future strategies to address these problems are proposed. These include increasing the sample size in homogenous ethnic groups, high throughput genotyping and genomewide linkage disequilibrium (LD) mapping to establish disease associated ancestral haplotypes. Elucidation of the function of particular genes ('functional genomics') in the pathogenesis of T1D will be a most important element in future studies in this field, in addition to more sophisticated methods of statistical analyses.

Genetic susceptibility to fibrocalculous pancreatic diabetes in Bangladeshi subjects: a family study.

Fibrocalculous pancreatic diabetes (FCPD) is an uncommon cause of diabetes, seen mainly in developing countries. A family-based study was carried out in 67 Bangladeshi families, consisting of a proband with FCPD and both parents, to determine whether an association exists between FCPD susceptibility and either the major histocompatiblity complex (MHC) or insulin gene (INS) loci. HLA-DQB1 typing was done using allele-specific primers, and INS was typed using the restriction enzyme HphI. Three microsatellites (TNFa, TNFc and TNFd), from within and flanking the TNF-LT locus, were used for MHC Class IV typing and a PCR-RFLP assay was used to define the -308G/A TNF promoter polymorphism. The extended transmission disequilibrium test (ETDT) was used for statistical analysis. An overall association was observed between FCPD and HLA-DQB1 (P = 0.003), that was largely due to a positive association with HLA-DQB1*0302 and a negative association with HLA-DQB1*0202. Although no association was found between FCPD and TNF-LT microsatellite markers a trend was observed for TNFc (P = 0.037, Pc = 0.15). No association was found between FCPD and INS (P = 0.26). This study confirms an association between FCPD and the MHC using a family-based study design and the stringent ETDT analysis; a novel protective association was found with HLA-DQB1*0202 in Bangladeshi FCPD subjects. The genetic susceptibility to FCPD has features both similar and dissimilar to T1DM.

Hereditary periodic fever syndromes.

Hereditary periodic fever syndromes are defined by recurrent attacks of generalised inflammation for which no infectious or auto-immune cause can be identified. For most of these disorders, the molecular basis has recently been elucidated. This has opened the prospect of novel therapeutic approaches. Familial Mediterranean fever (FMF) is caused by mutations in the MEFV gene. Pathogenesis is poorly understood. The clinical severity is in part related to the mutations involved. Tumour necrosis factor receptor-1-associated periodic syndrome (TRAPS) is caused by mutations in the TNFRSF1A gene. This results in decreased serum levels soluble TNF-receptor leading to inflammation due to unopposed TNF-alpha action. Results of treatment with recombinant TNF-receptor analogues are promising. The hyper IgD periodic fever syndrome (HIDS) is caused by mutations in the MVK gene, leading to mevalonate kinase deficiency. The pathogenesis remains unclear. Muckle-Wells syndrome (MWS) and familial cold urticaria (FCU) are probably allelic disorders. The gene has been located, but not identified.

TNF and TNFR biology in health and disease.

Many insights have been gained into cytokine-regulated control of inflammatory processes and host defence in recent years. Evidence has also gradually accumulated that cytokine cascades play a central role in events regulating cell death and differentiation. Further developments include an understanding that the biological effects of the tumor necrosis factor-alpha (TNF-alpha or TNFSF) cytokine may be regulated by soluble TNF receptor binding and that modulation of receptor levels may permit physiological inhibition of TNF action. There has been a gradual realisation of the value of TNF/TNFR ratios as predictors of disease outcome, and the discovery of functional regulatory polymorphisms of the TNF gene and mutations of TNFRSF1A (TNFR1 receptor) have led to conceptual breakthroughs in our understanding of the genetic control of inflammation. However the exact mechanisms by which TNFRSF1A mutations give rise to disease susceptibility are not yet well understood. Over the past 10 years these concepts have been used as the basis for successful anti-TNF therapy of autoimmune diseases like rheumatoid arthritis (RA) and Crohn's disease.

Tumour necrosis factor 5' promoter single nucleotide polymorphisms influence susceptibility to rheumatoid arthritis (RA) in immunogenetically defined multiplex RA families.

Tumour necrosis factor (TNF) plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA) and it has been shown that the TNF-lymphotoxin (TNF-LT) region influences susceptibility to RA. To investigate the role of the TNF-LT locus further, inheritance of TNF 5' promoter alleles was determined in multiplex RA families. Six previously defined TNF promoter single nucleotide polymorphisms (SNPs) (-238, -308, -376, -857, -863, -1031) were observed in these families and in addition, a heretofore undocumented adenine (A) to cytosine (C) substitution at position -572 relative to the transcription start site was defined. TNF 5' promoter SNPs were found to co-segregate with specific TNF microsatellite haplotypes. In particular, the SNP -308A allele was found to be inherited with the TNF a2, b3, c1, d1, e3 (H2) microsatellite haplotype (P < 0.001) which had previously been found to be associated with RA in individuals heterozygous for the HLA-DR 'shared epitope' (SE). When the data were stratified by the presence of the SE with further stratification according to SE DR subtypes and analysed by transmission disequilibrium test (TDT) for which offspring were assumed independent, the -308A and -857T alleles were found to be associated with RA in patients carrying the SE (P = 0.0076 and 0.0063 respectively). The data were further stratified to analyse for association in individuals homozygous or heterozygous for SE alleles. Results showed that the -308A allele was significantly associated with RA susceptibility in individuals heterozygous for the SE (P < 0.001) with the significance only occurring in patients carrying HLA-DR4 (P < 0.001), while the -857T allele was significant in individuals homozygous for the SE (P = 0.0039). Further analysis using the pedigree disequilibrium test (PDT) which conservatively adjusts for all sources of familial correlation except that conferred by linkage disequilibrium still indicated a significant role for the -308A and -857T alleles. These data provide evidence that TNF promoter SNPs may play an independent role in RA susceptibility in specific immunogenetically-defined groups of RA patients.