Philip L. Riches

SLC2A9 is a newly identified urate transporter influencing serum urate concentration, urate excretion and gout

Uric acid is the end product of purine metabolism in humans and great apes, which have lost hepatic uricase activity, leading to uniquely high serum uric acid concentrations (200–500 μM) compared with other mammals (3–120 μM). About 70% of daily urate disposal occurs via the kidneys, and in 5–25% of the human population, impaired renal excretion leads to hyperuricemia. About 10% of people with hyperuricemia develop gout, an inflammatory arthritis that results from deposition of monosodium urate crystals in the joint. We have identified genetic variants within a transporter gene, SLC2A9, that explain 1.7–5.3% of the variance in serum uric acid concentrations, following a genome-wide association scan in a Croatian population sample. SLC2A9 variants were also associated with low fractional excretion of uric acid and/or gout in UK, Croatian and German population samples. SLC2A9 is a known fructose transporter, and we now show that it has strong uric acid transport activity in Xenopus laevis oocytes.

Proteins secreted by the parasitic nematode Nippostrongylus brasiliensis act as adjuvants for Th2 responses

Infections with parasitic helminths such as Nippostronglyus brasiliensis induce dominant type 2 responses from antigen‐specific T helper cells. The potency of the Th2 bias can also drive Th2 responses to bystander antigens introduced at the same time as infection. We now report that the Th2‐promoting effect of infection can be reproduced with soluble N. brasiliensis excretory‐secretory proteins (NES) released by adult parasites in vitro. Immunization of BALB / c mice with NES results in the production of IL‐4 with elevated total serum IgE and specific IgG1 antibodies. NES is also able to stimulate IL‐4 and polyclonal IgE production in other mouse strains (C57BL / 6, B10.D2, CBA). These features are seen whether NES is administered without adjuvant as soluble protein in phosphate‐buffered saline or with complete Freunds adjuvant which normally favors Th1 responses. Thus, NES possesses intrinsic adjuvanticity. Moreover, co‐administration of hen egg lysozyme (HEL) with NES in the absence of other adjuvants results in generation of HEL‐specific lymphocyte proliferation, IL‐4 release and IgG1 antibody responses, documenting that NES can act as an adjuvant for third‐party antigens. Proteinase K digestion or heat treatment of NES before immunization abolished the IL‐4‐stimulating activity, indicating that the factors acting to promote Th2 induction are proteins secreted by the adult parasite.

Osteoporosis Associated with Neutralizing Autoantibodies against Osteoprotegerin

Autoantibodies against osteoprotegerin, which block the inhibitory effect of osteoprotegerin on signaling by the receptor activator of nuclear factor (NF)-kappaB (RANK), were identified in a man with celiac disease who presented with severe osteoporosis and high bone turnover. The osteoporosis did not respond to the treatment of his celiac disease but was completely reversed by bisphosphonate therapy. Autoantibodies against osteoprotegerin were detected in three additional patients with celiac disease. Such autoantibodies may be associated with the development of high-turnover osteoporosis, but whether autoantibodies against osteoprotegerin commonly contribute to the pathogenesis of osteoporosis in patients with celiac disease remains to be determined.

Recent insights into the pathogenesis of hyperuricaemia and gout

Gout is a common rheumatic disease in humans which is characterized by elevation in serum uric acid levels, and deposition of uric acid crystals in the joint. Hyperuricaemia is the primary risk factor for the development of gout and primates have uniquely high levels of serum uric acid due to missense mutations in the uricase gene. Levels of serum uric acid are known to be highly heritable, and mutations in genes which encode enzymes in the purine salvage pathway have long been recognized as rare causes of gout. Until recently, however, little has been known about the genetic determinants of urate metabolism and susceptibility to gout in the general population. Over recent months, a series of large scale genome wide association studies have been performed which have shed new light on the genes which regulate serum uric acid levels and susceptibility to gout. Most of these genes seem to be involved in regulating the renal excretion of uric acid which underscores the importance of reduced urate excretion as opposed to increased endogenous production as a cause of gout. Further work will now be required to investigate the mechanisms by which these genetic variants regulate urate excretion and serum urate levels. However, it seems likely that the genes so far identified will represent new molecular targets for the design of drugs to enhance urate excretion and the genetic variants that predispose to gout might be of value as genetic markers of susceptibility to gout.

Genetic variation in the **TNFRSF11A** gene encoding RANK is associated with susceptibility to Paget's disease of bone

RANK (receptor activator of nuclear factor‐κB), encoded by TNFRSF11A, is a key protein in osteoclastogenesis. TNFRSF11A mutations cause Pagets disease of bone (PDB)–like diseases (ie, familial expansile osteolysis, expansile skeletal hyperphosphatasia, and early‐onset PDB) and an osteoclast‐poor form of osteopetrosis. However, no TNFRSF11A mutations have been found in classic PDB, neither in familial nor in isolated cases. To investigate the possible relationship between TNFRSF11A polymorphisms and sporadic PDB, we conducted an association study including 32 single‐nucleotide polymorphisms (SNPs) in 196 Belgian sporadic PDB patients and 212 control individuals. Thirteen SNPs and 3 multimarker tests (MMTs) turned out to have a p value of between .036 and 3.17 × 10−4, with the major effect coming from females. Moreover, 6 SNPs and 1 MMT withstood the Bonferroni correction (p < .002). Replication studies were performed for 2 nonsynonymous SNPs (rs35211496 and rs1805034) in a Dutch and a British cohort. Interestingly, both SNPs resulted in p values ranging from .013 to 8.38 × 10−5 in both populations. Meta‐analysis over three populations resulted in p = .002 for rs35211496 and p = 1.27 × 10−8 for rs1805034, again mainly coming from the female subgroups. In an attempt to identify the underlying causative SNP, we performed functional studies for the coding SNPs as well as resequencing efforts of a 31‐kb region harboring a risk haplotype within the Belgian females. However, neither approach resulted in significant evidence for the causality of any of the tested genetic variants. Therefore, further studies are needed to identify the real cause of the increased risk to develop PDB shown to be present within TNFRSF11A.

Prevalence and clinical prediction of osteoporosis in a contemporary cohort of patients with rheumatoid arthritis

OBJECTIVE Osteoporosis has previously been reported to be twice as common in patients with RA as in controls, but these studies predate the introduction of aggressive management of RA. The aim of this study was to evaluate the prevalence and clinical predictors of osteoporosis in RA in a contemporary cohort and to develop a clinical tool for the identification of patients at risk. METHODS The prevalence of osteoporosis was related to clinical and demographic variables in 304 consecutive RA patients undergoing DXA at a single centre between 2009 and 2010 and compared with the frequency of osteoporosis in a population-based cohort of 903 subjects. RESULTS The RA cohort was predominantly female (81.9%), with an average age of 63.5 years (s.d. 11.8) and a disease duration of 9.6 years (s.d. 10.2). Osteoporosis was present in 91 (29.9%) patients at either the spine or total hip compared with 157/903 (17.4%) of age- and gender-matched controls. In RA patients, osteoporosis was associated with female gender (P = 0.002), age (P < 0.001), time since menopause (P < 0.001), BMI (P < 0.001), ESR (P = 0.006), Larsen score (P = 0.011) and co-morbidities (P = 0.020), but logistic regression analysis showed that only age and BMI were independent predictors. A predictive tool based on age and BMI was developed that had 91.4% sensitivity for the detection of osteoporosis in an independent RA population. CONCLUSION The prevalence of osteoporosis in RA remains high in the modern era despite aggressive management and the use of biologic therapy. Most RA patients with osteoporosis can be identified by a simple algorithm taking age and BMI into account.

Multiplicative interaction of functional inflammasome genetic variants in determining the risk of gout.

IntroductionThe acute gout flare results from a localised self-limiting innate immune response to monosodium urate (MSU) crystals deposited in joints in hyperuricaemic individuals. Activation of the caspase recruitment domain-containing protein 8 (CARD8) NOD-like receptor pyrin-containing 3 (NLRP3) inflammasome by MSU crystals and production of mature interleukin-1β (IL-1β) is central to acute gouty arthritis. However very little is known about genetic control of the innate immune response involved in acute gouty arthritis. Therefore our aim was to test functional single nucleotide polymorphism (SNP) variants in the toll-like receptor (TLR)-inflammasome-IL-1β axis for association with gout.Methods1,494 gout cases of European and 863 gout cases of New Zealand (NZ) Polynesian (Māori and Pacific Island) ancestry were included. Gout was diagnosed by the 1977 ARA gout classification criteria. There were 1,030 Polynesian controls and 10,942 European controls including from the publicly-available Atherosclerosis Risk in Communities (ARIC) and Framingham Heart (FHS) studies. The ten SNPs were either genotyped by Sequenom MassArray or by Affymetrix SNP array or imputed in the ARIC and FHS datasets. Allelic association was done by logistic regression adjusting by age and sex with European and Polynesian data combined by meta-analysis. Sample sets were pooled for multiplicative interaction analysis, which was also adjusted by sample set.ResultsEleven SNPs were tested in the TLR2, CD14, IL1B, CARD8, NLRP3, MYD88, P2RX7, DAPK1 and TNXIP genes. Nominally significant (P < 0.05) associations with gout were detected at CARD8 rs2043211 (OR = 1.12, P = 0.007), IL1B rs1143623 (OR = 1.10, P = 0.020) and CD14 rs2569190 (OR = 1.08; P = 0.036). There was significant multiplicative interaction between CARD8 and IL1B (P = 0.005), with the IL1B risk genotype amplifying the risk effect of CARD8.ConclusionThere is evidence for association of gout with functional variants in CARD8, IL1B and CD14. The gout-associated allele of IL1B increases expression of IL-1β – the multiplicative interaction with CARD8 would be consistent with a synergy of greater inflammasome activity (resulting from reduced CARD8) combined with higher levels of pre-IL-1β expression leading to increased production of mature IL-1β in gout.

The toll-like receptor 4 (TLR4) variant rs2149356 and risk of gout in European and polynesian sample sets

Deposition of crystallized monosodium urate (MSU) in joints as a result of hyperuricemia is a central risk factor for gout. However other factors must exist that control the progression from hyperuricaemia to gout. A previous genetic association study has implicated the toll-like receptor 4 (TLR4) which activates the NLRP3 inflammasome via the nuclear factor-κB signaling pathway upon stimulation by MSU crystals. The T-allele of single nucleotide polymorphism rs2149356 in TLR4 is a risk factor associated with gout in a Chinese study. Our aim was to replicate this observation in participants of European and New Zealand Polynesian (Māori and Pacific) ancestry. A total of 2250 clinically-ascertained prevalent gout cases and 13925 controls were used. Non-clinically-ascertained incident gout cases and controls from the Health Professional Follow-up (HPFS) and Nurses Health Studies (NHS) were also used. Genotypes were derived from genome-wide genotype data or directly obtained using Taqman. Logistic regression analysis was done including age, sex, diuretic exposure and ancestry as covariates as appropriate. The T-allele increased the risk of gout in the clinically-ascertained European samples (OR = 1.12, P = 0.012) and decreased the risk of gout in Polynesians (OR = 0.80, P = 0.011). There was no evidence for association in the HPFS or NHS sample sets. In conclusion TLR4 SNP rs2143956 associates with gout risk in prevalent clinically-ascertained gout in Europeans, in a direction consistent with previously published results in Han Chinese. However, with an opposite direction of association in Polynesians and no evidence for association in a non-clinically-ascertained incident gout cohort this variant should be analysed in other international gout genetic data sets to determine if there is genuine evidence for association.

Differential effects of bacterial lipopolysaccharides upon neutrophil function.

Lipopolysaccharide (LPS) is a potent inflammatory agent which augments neutrophil sensitivity to subsequent inflammatory stimuli. In this study, the effects of structurally different LPS types upon neutrophil effector functions were examined. Rough LPS types, which have lost the O‐polysaccharide moiety, were found to act more rapidly than smooth LPS types in stimulating neutrophil β2 integrin activity and fMLP‐induced respiratory burst. These findings suggest an involvement of the O‐polysaccharide region of LPS in regulating neutrophil responsiveness to different LPS chemotypes with important implications for the mechanisms underlying regulation of the inflammatory response in conditions associated with elevation of LPS in plasma, e.g. septic shock or acute respiratory distress syndrome.

Replication of association of the apolipoprotein A1-C3-A4 gene cluster with the risk of gout

OBJECTIVE Gout is associated with dyslipidaemia. Association of the apolipoprotein A1-C3-A4 gene cluster with gout has previously been reported in a small study. To investigate a possible causal role for this locus in gout, we tested the association of genetic variants from APOA1 (rs670) and APOC3 (rs5128) with gout. METHODS We studied data for 2452 controls and 2690 clinically ascertained gout cases of European and New Zealand Polynesian (Māori and Pacific) ancestry. Data were also used from the publicly available Atherosclerosis Risk in Communities study (n = 5367) and the Framingham Heart Study (n = 2984). Multivariate adjusted logistic and linear regression was used to test the association of single-nucleotide polymorphisms with gout risk, serum urate, triglyceride and high-density lipoprotein cholesterol (HDL-C). RESULTS In Polynesians, the T-allele of rs670 (APOA1) increased (odds ratio, OR = 1.53, P = 4.9 × 10(-6)) and the G-allele of rs5128 (APOC3) decreased the risk of gout (OR = 0.86, P = 0.026). In Europeans, there was a strong trend to a risk effect of the T-allele for rs670 (OR = 1.11, P = 0.055), with a significant protective effect of the G-allele for rs5128 being observed after adjustment for triglycerides and HDL-C (OR = 0.81, P = 0.039). The effect at rs5128 was specific to males in both Europeans and Polynesians. Association in Polynesians was independent of any effect of rs670 and rs5128 on triglyceride and HDL-C levels. There was no evidence for association of either single-nucleotide polymorphism with serum urate levels (P ⩾ 0.10). CONCLUSION Our data, replicating a previous study, supports the hypothesis that the apolipoprotein A1-C3-A4 gene cluster plays a causal role in gout.