G-CAN 2015 Inaugural Scientific Meeting

November 6, 2015

San Francisco, California USA

SUMMARY

Introduction to G-CAN

The mission of G-CAN, a 501C3 nonprofit organization conceived in 2014, and formed in January 2015, is to

develop globally networked multidisciplinary collaborative and networked new projects and initiatives to fulfill

an array of unmet needs in the areas of gout research with a focus on:

Gout pathophysiology, diagnostic approaches, prognostic and response markers, treatment targets,

outcomes, and quality of care

Hyperuricemia and its connection to cardiovascular, metabolic and other co-morbidities

Other crystal arthropathy: focus on articular CPPD and BCP crystal deposition disorders with and

without osteoarthritis

Developing, testing, and validating new models of patient and provider education

Fostering the recruitment and development of new investigators in these fields

Organizational sponsors function as partners, galvanizing projects. G-CAN’s founding support was provided by

ARDEA Biosciences/AstraZeneca. Sponsors subsequently committed to G-CAN are Takeda, SOBI, Crealta

Pharmaceuticals, LLC, and CymaBay Therapeutics.

This is a summary of the Inaugural Scientific Meeting of G-CAN, on November 6, 2015, with five sessions

being held, in the format of 7 slides/10 minutes “lightning talks”, including participation by and interaction with

our G-CAN sponsors as partners.

Session on G-CAN Lead Project 1

International Gout Database Development

Database Genetics and Environmental Factors. Genome-Wide Association Studies and Next

Generation Genetics

Tony Merriman, PhD, University of Otago, New Zealand

Problem 1: GWAS in clinically-ascertained gout relatively small

Very large genome-wide association studies (GWAS) are the bedrock for knowledge advances. However

GWAS from clinically ascertained gout are relatively small.

There is a good knowledge base from previous studies: A European study by Kottegen et al using more than

140,000 participants culled from existing studies and some earlier urate-control GWAS have identified 28 loci.

From this, new knowledge on uric acid transporters was obtained, but offered no insights about molecular

control.

There have been a few GWAS in clinically ascertained gout:

A Chinese GWAS study by Li et al in 2015 of 1,255 cases found 1 significant locus and 3 novel loci.

A Japanese study by Matsuo et all in 2015 looked at 945 cases and found 3 significant loci and 2 novel

loci.

An international collaboration between Eurogout, Australasia and the US looked at 5,000 cases.

However when compared to rheumatoid arthritis studies for example, these are still relatively small. A

rheumatoid arthritis study by Okada et al in 2014 looked at 29,880 cases resulting in a total of 101 loci and a

discovery of 42 novel loci.

Using the summary level statistics from existing gout data, there is a limited amount of sub-analyses.

We decided early on that we needed to recruit cases in gout and that it needs to be largely hospital based. The

Eurogout and European Crystal Network led by Alex So and Fred Liote is a good example of the kind of

genetics recruitment project that is needed to obtain better data. The European Crystal Network uses mostly

hospital based recruitment from:

Germany

Ireland

Spain

Switzerland

The Netherlands

United Kingdom

There is a commitment of time and resources from contributors for things such as ethics, recruitment and data

collections. The DNA is either prepared locally and sent to New Zealand or whole blood is sent.

Problem 2. Under-utilization of publicly-available genetic databases of individual level data

There are databases already in existence that could be utilized to address important questions such as the

genetic basis of urate control and gout, causal relationships by Mendelian randomization and environmental

interactions. There are a lot of environmental dietary databases not used widely enough but that could be very

useful analytically and for posing research questions.

Examples of databases with rich phenotype and environmental data include dbGaP:

ARIC/FHS/CHS/JHS/CARDIA (10,000s for urate), the Kaiser Permanente (~100,000 for gout and urate) and

the UK Biobank (500,000 for gout and urate).

These databases are underutilized in part because of barriers such as the application and approval process,

the need to compute infrastructure and database management and the analytical and statistical skills needed

to pose and address research questions that make the data useful.

Another reason there have not been enough GWAS studies are because gout research has been historically

underfunded and “unfashionable.” The demographics of gout are working-aged men with intermittent problems,

and the perception is that it is a life-style issue. Because of this, there are not enough clinical resources allotted

to its study and there are too few researchers – particularly new, young ones.

Solutions Proposed solutions would be to stimulate G-CAN to do something similar to the European study; meta-analyze and/or poll the existing clinically ascertained GWAS data sets. This can immediately be done by collaboration.

Additionally we need to facilitate access to publicly available genetic data sets, removing barriers to answering important questions. An immediate resource for short-term projects is needed as short-term projects are a good way of attracting new researchers and emerging investigators.

If we are going to increase GWAS from clinically ascertained gout and broaden sub-analyses we need international recruitment for new gout genetics databases in the tens of thousands from primary care. Also, clinically based genetic recruitment should be routine at clinical trials. This approach can be applied to other crystal pathologies. Extensive data collection will be required as well as a new powerful resource for short term projects.

These solutions would provide genome sequencing (next generation genetics) and discovery of rarer variants of higher penetrance for functional studies. They would also allow individual genetic analysis of a very large database.

Proposal

Our proposal for G-CAN is facilitation of access to publically available genetics data sets and new international

multi-ancestral gout genetics databases as related G-CAN projects.

To implement this, a steering group of 10 to 15 people should initially be established with international

representation, including geneticists with experience in other phenotypes (e.g. rheumatoid arthritis); clinical

researchers, and those from primary care and with database expertise. Currently confirmed for this steering

committee are: Hyon Choi (US), Tony Merriman and Nicola Dalbeth (New Zealand), Alex So (Switzerland), Eli

Stahl (US), and Robert Terkeltaub (US).

Functional Genetics – ABCG2, SLC2A9 Experience, GLUT9 and Hyperuricemia David Mount, MD FRCPC Harvard Medical School

Five previous GWA studies initially reported an effect on serum urate of common variants in GLUT9, a fructose

transporter encoded by SLC2A9. This is a modest effect, with a change of about 3.1 mg/dl, but it is modified by

body mass index and gender.

Variation in SLC2A9 genotype accounts for 1.2% of the variance in uric acid in men and 6% in women, making

it the single biggest gene effect found so far in gout. Associated single nucleotide polymorphisms (SNPs) are

scattered throughout the gene; the causal variants have not been characterized.

This is an instance when functional genetics can help to better understand the role SLC2A9 and GLUT9 play in

hyperuricemia. To understand the genetics behind this mechanism requires going “back to the bench.”

Proximal urate absorption is regulated by transport of urate in the renal proximal tubules. GLUT9 is the

mechanism for reabsorption of urate (large amounts of GLUT9 are found in uric acid secretion fractions). We

know that SLC2A9 urate transport is activated by membrane depolarization and inhibited by uricosurics, such

as benzbromarone. GLUT9 exists as two alternatively spliced variants; GLUT9a and GLUT9b. GLUT9b which

transports uric across the apical membrane, has been found to be activated by insulin.

The question posed is, “What is turning the gene off and on?” It is unusual to understand the mechanisms for a

gene to be turned on or off and a phenotype created. There is at least one inhibitor for GLUT9 function.

One study used a membrane yeast two-hybrid system to look for clues as to what is turning on resistance or

reporter genes.

Another study looked at the effect of ITM2B co-expression on GLUT9 in Xenopus oocytes. Oocytes are a

good model for insulin dose-dependent activation of GLUT9b. Frog eggs were microinjected with cRNA and

uric acid uptake was observed for over an hour. The investigators found that if you then depolarize the cell,

there is a huge activation inhibited by benzbromarone.

How is it regulated? We know that GLUT9b has 7 different promoters. Looking at the relative activity of

SLC2A9 promoters, we find that GLUT9A is potent promoter and GLUT9b is weak.

If you look at structure function studies of how GLUT9 works and the functional effects of coding cSNPs you

see that depolarized cells rR265H have no function under non-polarized but it can be can turned on by

switching intracellular loops.

What is needed is a careful uptake experiment to see how the transporter works.

There is no known cause of mutation in GLUT9. We are still trying to understand the structure. Studies of

GLUT9 and GLUT9a and 9b have greatly expanded structure of the gene and there are 28 known GWAS

genes for urate identified and more to come.

It is a major advance to have a huge laundry list of genes. But we still don’t have the common SNPs to know if

it is the right gene. There are a number of orphan genes with unknown functions.

Why is it important to focus on GLUT 9? If we were to pick one transporter that was key to urea absorption this

would be the one. GLUT9 is relevant to metabolic syndrome – it is part of the insulin-regulated GLUT family of

transporters and it is the exclusive exit pathway for urate in renal reabsorption. Perhaps there could be a major

final common pathway in urate homeostasis?

Also GLUT9 is a testable interactor for other hyperuricemic genes. There is a major gene effect in

hyperuricemia with uncharacterized causative SNPs – in other words, this is a major gene effect where we

don’t understand the mechanism.

Asian GWAS Databases for Uric Acid/Gout and the RA Experience Yukinori Okada Department of Human Genetics and Disease Diversity, Tokyo Medical and Dental University

Laboratory for Statistical Genetics, IMS, RIKEN

Integration of data from GWAS and biological studies could lead to a better understanding of gout mechanisms

and drug treatments, as has been the case in rheumatoid arthritis.

A GWAS meta-analysis for serum uric acid in East Asians was conducted by the AGEN consortium in 2012. It

was not a big sample size (n = 33,047 for the GWAS + replication study). The study identified 3 known genetic

loci (SLC2A9, ABCG2, SLC22A12) and 1 novel genetic locus (MAF). Another GWAS of clinically defined gout

in Japanese, published by Matsuo et al in the Ann Rheum Dis this year, identified 5 loci.

We devised an in silico pipeline using established bioinformatics methods based on functional annotation, cis-

acting expression quantitative trait loci and pathway analyses — as well as novel methods based on genetic

overlap with human primary immunodeficiency, haematological cancer somatic mutations and knockout mouse

phenotypes — to identify 98 biological candidate genes at these 101 risk loci.

The integration of genetics with biological and medical “big data” should help us to understand disease biology

and drug discovery. This integration model has been used successfully in RA drug development

Looking at explained heritability in European and Asian populations, causality of RA risk genes/variants could

be annotated as:

(1) Missense SNPs and immune cell specific cis-eQTLs,

(2) Epigenetic histone marks (H3K4me3) in regulatory T cells.

(3) Overlap with hematological cancer somatic mutation genes and GWAS risk loci

The integration of GWAS and miRNA target-gene networks in RA showed significant impact of miRNA on the

genetics of human disease. Our method provides a list of miRNA target genes in RA that could be promising

for drug discovery and drug repurposing. Such a list could be similarly useful for gout.

Challenges of Integrating Big Databases: Environmental Factors and EWAS

Eli Stahl, PhD Icahn School of Medicine at Mt. Sinai

What are opportunities in front of us?

We need the kinds of “big data” that come from large-scale GWAS, e.g. the Asian GWAS for urea: Where is

the Asian European meta-analysis? To move forward, we need data from:

• Integrated analyses with plethora of genetic data available pathways etc. e.g. Uric GWAS expression

profile data from post mortems Phantom 5 data. Large-scale GWAS (N, NSNPs)

• Consortia, Ethnicity, Phenotypes

• Functional genomic data

• Pathways, Networks

• Expression profiles, Chromatin states, Cell types

• Phenotypic data

• Prospective/Longitudinal cohorts

• EMR cohorts

The applications from these kinds of data can help answer important questions.

Application 1. Causes of Gout. Urate GWAS can provide information on the mechanisms and pathways of

urate production.

Application 2. Gout-specific effect sizes. Genetic studies with normouricemic controls that help determine

thresholds for increased risk of the disease, similar to what has been done in genetic studies of schizophrenia

risk and genetic overlap.

Application 3. Pleiotropy/Secondary phenotypes. Studies to identify genetic variants that could influence the

risk of gout.

The “dream” application of big data is “Deep Knowledge of Target Pathways.” This would be using the existing

data to identify:

• Candidate pathways and targets

• Direction of effect, mechanism of action

• Causation, Mendelian randomization

• Drug targets, repurposing

• Secondary phenotypic effects

Gout research needs to catch up with what has been done in studies of other inflammatory diseases and to link

up inflammatory disease, kidney function, and metabolic disease. G-CAN members need to see this challenge

as an opportunity to find creative applications of statistical genetics data such as EHR cohorts, PheWAS and

EWAS.

Session on G-CAN Lead Project 2

Hyperuricemia and Cardiovascular-Renal Disease: Multicenter Collaborative

Project Development

Pathophysiology: Uric Acid and End-organ Liver and Renal Damage Daniel I. Feig, MD, PhD Director, Division of Nephrology Department of Pediatrics

University of Alabama, Birmingham

Are elevated Uric acid levels associated with decline in kidney function and Non-Alcoholic Fatty Liver Disease?

An Italian study looked at men and women with normal renal function and serum uric acid levels of 6 and 7

mg/dl. This is not a high risk but over 3 years, it accounted for the development of chronic kidney disease. This

study shows that in both men and women, a higher quartile of serum uric acid is associated with greater GFR

decline in subjects with normal renal function. There are 14 published studies that show similar results in

patients with CKD at the start of the study. The relationship is robust across CKD1-4.

Another study looked at several thousand people, all obese, for an association with NAFLD. The prevalence of

NAFLD was higher in those participants that had higher SUA levels, adjusting for multiple factors, including

metabolic syndrome. The study concluded that individuals with hyperuricemia were more likely to have NAFLD

have elevated liver enzymes.

Uric acid is vasculopathic, resulting in vasoconstriction and altered compliance in resistance vessels and organ

perfusion.

The presence of elevated uric acid leads to a two-step change in blood vessels. The first phase is reversible

vasoconstriction and the second arterial wall thickening. In the first phase, Uric acid leads to increased renin

and decreased nitric oxide. The vessel remains uric acid-dependent and sodium resistant.

In the second phase, there is vascular smooth muscle proliferation mediated by PDGF and MCP-1. The vessel

becomes uric acid-independent and sodium sensitive.

Uric acid passes through the membrane leading to a cascade of kinases etc, released in narrowing of vessels.

This may be the crux of progressive renal damage and liver damage.

The mechanism for NAFLD is also related to the vasculopathic effects of hyperuricemia as well as insulin

resistance leading to fat deposition in hepatocytes. This activation to leads to cirrhosis.

Understanding these mechanisms provides molecular targets for dietary intervention, uric acid production or

clearance and mediated mechanisms.

Therapeutic Potential of Xanthine Oxidase Inhibition as an Inhibitor of Atherogenesis

Xanthine oxidase: Old Dog New Tricks? Alex So, MD CHUV and University of Lausanne

Hyperuricemia crosses boundaries and involves many different organs – renal, cardiac and liver. It can be can

be reversible or irreversible. Xanthine oxidase (XO) plays a role in inflammation and the immune response.

Inflammation is a crucial component of hyperuricemia and co-morbidities and XO is also important in terms of

regulation of this inflammation.

Xanthine oxidoreductase (XOR) is found in two forms: Constitutively as Xanthine dehydrogenase (XDH) and

under oxidative stress conditions as XO. The XO form is the predominant one during an immune response. We

know that XDH can be converted to XO in two ways, proteolytic cleavage or S-S bond modification. In this

pathway hypoxanthine converts to xanthine and to uric acid.

XO inhibition can reduce uric acid, inflammation and other associated morbidities. Looking at what we know

from treatment studies of liver and cardiovascular inflammation, Allopurinol and Febuxostat are effective in

inhibiting XO and thereby lowering the uric acid effect and inflammation. Studies have also looked at how

siRNA and other knockouts can affect the inflammatory effect. There are two types of knockouts in mice

pathways to be investigated.

In vitro, BMDM stimulation studies and in vivo crystal induced peritonitis studies demonstrate how crystals

induce XO and Il-1β release to cause inflammation. XO inhibition blocks crystal-induced ROS formation and IL-

1β release. If you block the metabolisms it doesn’t differentiate between the effect. XO inhibition only inhibits

NLRP3 inflammasome activation.

Where is XO located in the cell? XOR activity is found both in the mitochondria and the cytoplasm. XO could

exist in both compartments.

In vivo, if you treat mice with Febuxostat, you get reduction of atherosclerosis and the number of cells with

crystals decreases. In vitro, Febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory

cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-

mediated ROS formation is pro-inflammatory and XO inhibition by Febuxostat is a potential therapy for

atherosclerosis. Febuxostat reduces experimental atherosclerosis in mice.

Trials with Febuxostat suggest that it is possible to intervene in this process. In three trials 53 to 158 patients

with chronic kidney disease were given Febuxostat. Over several months the progression of a portion of

population from one level of chronic kidney disease to next was reduced by 20% and vasodilatation was

improved.

Research agenda: Specific areas to investigate in XO inhibition would be:

o We need new agents. Is XO inhibition beneficial in other clinical conditions, i.e. does XO

inhibition have a role in other inflammatory pathways?

o How low can UA be before causing a problem?

o Novel agents for selectively blocking XO form.

o Molecular mechanisms of XO ROS activity

Observational Studies and Mendelian Randomization Hyon Choi, MD Professor of Medicine Harvard Medical School Massachusetts General Hospital

Gout and its causal precursor, serum uric acid (SUA), have been associated with many cardiovascular (CV)-

metabolic-renal outcomes, including an increased risk of coronary artery disease (CAD), stroke, type 2

diabetes, insulin resistance syndrome, chronic kidney disease (CKD), and hypertension. However, due to

confounding and reverse causation, it is difficult to establish causality in epidemiology.

Genetic association can be used to infer causality of a biomarker with a disease endpoint. This approach,

called Mendelian Randomization, infers causality because an individual’s genotype is assigned randomly at

meiosis, thereby eliminating bias by confounding variables and reducing spurious findings in observational

epidemiology. Using gene variants that affect a biomarker of interest, we have confirmed low-density

lipoprotein cholesterol (LDL-C) as a causal risk factor for CAD and have refuted a causal role of high-density

lipoprotein cholesterol (HDL-C) (Voight et al. Lancet, 2012). Another prominent example is debunking a causal

role of CRP, which attracted a tremendous level of clinical and research interest over the past decade (Zacho,

et al. NEJM, 2009). (Our recent analyses also confirmed this non-causal relationship of CRP for the risk of

CAD.)

Furthermore, we have recently developed and implemented a modified approach to Mendelian Randomization,

called Multi-Phenotype Mendelian Randomization (MPMR), to isolate causal influences among a set of

correlated CV-metabolic risk factors for CAD (Do et al. Nature Genetics. 2013). This approach leverages

estimates of the effect sizes of genetic variants obtained from a meta-analysis of association results from a

genome-wide association study (GWAS) for CV-metabolic risk factor traits and CAD. For example, using a

model accounting for effects on LDL-C and/or HDL-C, we determined that the strength of a polymorphism's

effect on triglyceride levels is correlated with the magnitude of its effect on CAD risk. These results provide

evidence that triglyceride-rich lipoproteins causally influence the risk for CAD (Do et al. Nature Genetics.

2013). The same analysis can be applied to examine the causality of SUA on the risk of CAD and related CV-

metabolic traits.

We are applying the Mendelian Randomization approach to determine whether SUA levels are causally related

to CAD/myocardial infarction (MI) and cardiovascular disease (CVD) risk factors. To achieve this goal, we are

leveraging a series of large-scale datasets that we have assembled: (1) data from a GWAS of SUA and gout in

>140,000 individuals; (2) data from a GWAS of CAD in >85,000 individuals; and (3) data from several GWASs

of 10 CV-metabolic risk factors for CAD (i.e., type 2 diabetes, insulin resistance, blood pressure, body mass

index [BMI], CRP, LDL-C, HDL-C, plasma triglycerides, fasting glucose, and lipoprotein(a)).

Interim results on three traits (BMI, systolic blood pressure, and gout) to date have shown no evidence of a

causal relationship between SUA and systolic blood pressure. In contrast, we observed the evidence that BMI

is casually associated with SUA (as was shown by previous studies). Furthermore, we confirmed that SUA is

causally related to gout (a positive control). We found no evidence for a causal relation from SUA to BMI or

systolic blood pressure to SUA.

These MR study findings could guide the decision to pursue costly trials of urate-lowering agents for these

major CV-metabolic target outcomes. Such studies could also lead to additional studies of the mechanisms

involved, providing new insights into uric acid metabolism and CV-metabolic conditions.

We are continuing our investigation to determine whether any causal effects of SUA levels on CVD are

mediated by the rest of the 10 CV-metabolic-renal correlates.

Intervention Studies 1 – Hypertension, Renal Uric Acid and Blood Pressure:

Cause or Coincidence?

John P. Forman, MD, MSc Harvard Medical School Brigham and Women’s Hospital

Questions remain unanswered about the relationship between high blood pressure and high UA.

Is high uric acid a cause of increased blood pressure and hypertension?

Does reducing uric acid lower blood pressure?

Should we treat asymptomatic hyperuricemia as a means to prevent hypertensive complications?

Previous studies have been mostly inconclusive;

Randomized trials

◦ Multiple trials of allopurinol

In most, BP is a secondary endpoint

In most, there is a high risk-of-bias studies

In most, no significant effect was found

One small pilot trial of inosine (raising uric acid levels) had no effect

The most rigorous published trial thus far looked at Allopurinol vs. Probenecid vs. placebo in adolescents. This

showed a very large effect on UA levels.

Mendelian randomization studies have so far had inconsistent findings

This leaves us with several unanswered questions;

Is high uric acid a cause of increased blood pressure and hypertension?

Does reducing uric acid lower blood pressure?

Should we treat asymptomatic hyperuricemia as a means to prevent hypertensive complications?

To answer these questions, we need a large-scale, long-term, low risk-of-bias trial of raising uric acid (inosine

for Parkinson’s) with 270 patients and a two year follow-up. Blood pressure should be rigorously monitored in

this study.

Additionally there should be a large scale, long-term low risk of bias trial of uric acid lowering conducted on

adolescents and adults, looking at prehypertension and mild hypertension and a broad range of uric acid levels

in a racially and ethnically diverse population.

Intervention Studies 2 – Cardiac Allan Struthers, MD University of Dundee

At high doses, xanthine oxidase inhibitors (XOI) improve endothelial/vascular function by decreasing OS and

not by decreasing urate.

Left ventricular hypertrophy is a culprit because it reduces coronary perfusion reserve, is intrinsically

arrhythmogenic, leading to diastolic dysfunction, heart failure and sudden deaths, as well as LA enlargement,

atrial fibrillation and cardioembolic strokes.

A study looked at patients with angina and the effect of allopurinol on the change in LVMI and LVM. Allopurinol

significantly reduced LVMI and LVM when compared with placebo after nine months of therapy. It concluded

that high-dose allopurinol regresses LVH, reduce LV end-systolic volume and improves endothelial function in

patients with IHD and LVH.

Allopurinol works by inhibiting XO. When you inhibit the XO you decrease uric acid production and superoxide

anion generation. This mechanism is unlike beta blockers, which offload the heart and decrease oxygen

consumption. It may be that because the heart is using less oxygen, it is doing less work and may be the

cause of decreasing MIs.

Randomized control trials with Allopurinol concluded that Allopurinol significantly reduced cardiac events.

Allopurinol as an anti-angina agent:

1. Allopurinol improves key CV surrogates

- Endothelial function

- Vascular oxidative stress

- LV Hypertrophy

- Ischemia in angina

1. Observational data mostly supportive that Allopurinol reduces CV events

2. Small randomised trials suggest Allopurinol reduces CV events (except in heart failure)

Session on G-CAN Lead Project 3

Improving Point of Care Diagnosis of Crystal Arthropathy and Assessment of

Body Urate Burden

Raman Spectrometry as a Screening Tool at Point of Care Ozan Akkus, PhD Case Western Reserve

The majority of gout patients are diagnosed and managed by a primary care provider. This is often a

presumptive diagnosis based on symptoms. However a definitive diagnosis of gout requires identification of

crystals in the joint. Polarized light microscopy is the most common method for this, but the microscope and

the certified technicians to use it are often absent in a primary care clinical setting.

Enabling fluid analysis at the point of care may enable wider diagnosis of gout at the primary care level and

help admission decisions in other settings.

One tool that might offer a solution is Raman analysis of synovial fluid. This method identifies materials through

chemical bonds unique to MSU or CPP. Although Raman Spectroscopy is 100% accurate, the lab-grade

version of it is also expensive and complex.

Our research looked at using a rendered Raman unique to MSU and CPP that can be performed in a primary

care setting.

In this procedure, synovial fluid is drawn and then prepared with reagents. It is then injected through a micro

filter cartridge and the filtrate put in a shoebox-size device connected to a laptop. The entire process takes less

than an hour and provides automated synovial fluid analysis without expert knowledge. This method provides

clinicians with an unbiased objective decision making tool using lower-cost components.

To evaluate the effectiveness of the POCR, a comparison was done with Microscopic Analysis on 174 patients

with symptomatic joints. The POCR was conducted at a lab. The compensated polarized microscopy was

performed by CLIA certified operators.

For MSU, the two methods had close agreement in 164/174 with a kappa coefficient of .84. For CPP there was

moderate agreement in 162/174 with a kappa coefficient of .61

This method can be calibrated to measure the concentration of crystals for MSU and CPP.

POCR can provide a solution to unmet needs such as enabling diagnosis at the point of primary care,

expediting the diagnosis and helping make admission decisions in urgent/emergency care. It also provides an

automated synovial fluid analysis without expert knowledge.

POCR has only been carried out in the research lab so far. True point of care application would require

software/hardware improvements, assessment by clinical staff and performance assessment in the by the

clinicians to demonstrate applicability.

Nonetheless POCR has many advantages including accurate and timely diagnosis allowing providers in the

primary care setting to diagnose or rule out gout, start treatment sooner. It also has the potential for use at

other clinical locations or outside health care settings.

Increasing Ease of Recognition and Classification of Basic Ca+ Phosphate Crystals in Osteoarthritis Hang-Korng Ea, MD Paris Diderot University

A number of crystals associated with inflammation can be found in synovial fluid including monosodium urate,

calcium pyrophosphate dihydrate and basic calcium phosphate (BCP)

These crystals are small and can’t be seen by light microscopy unless they are in clumps. This poses two

problems; first of all there are two different kinds of crystals and they can be difficult to detect.

BCP crystals are ffound in 60% of synovial fluids, 100% of OA meniscus and 100% of knee and hip OA

cartilages obtained at surgery. They correlate to destruction of the cartilage and a higher risk of radiographic

progression.

Being able to identify these crystals would provide new insights into OA pathogenesis and also new targets for

development of treatment. However there are several challenges in identifying them; their small size, the

presence of others calcium-containing crystals and proteins that make them difficult to see, the difficulty in

obtaining normal controls of synovial fluid and the stability of the crystals which is impacted by storage

conditions.

Current BCP crystal identification tools are divided into imaging methods and spectroscopy methods. Imaging

methods include light and polarized light microscopy with special stains and digital-contact radiography (DCR).

Both of these are non-specific and can result in false positives. SEM, TEM and AFM are more accurate but

expensive, require expertise and are not widely available. Fourier-transform infrared spectroscopy (FTIR) can

be destructive while Synchrotron FTIRR and Raman spectroscopy are expensive and require expertise skills.

Few library spectra are available.

X-ray diffraction is also another way to identify these crystals but the samples must be pure.

Routine tools such as X-ray, CT scan or DCR can be used to enhance BCP crystal detection. However they do

not differentiate calcium-containing crystals. DCR is not applicable in whole joint samples.

Another method is to centrifuge the synovial fluid and analyze the pellet with light microscopy and dyes.

Alizarin red, oxytetracycline, Fluo-4 and bisphosphonate bind indifferently to calcium-containing crystals. There

is a high false positive rate with this method and the sensitivity depends on dye concentration, temperature,

and incubation time. Detection and extraction can also be performed with magnetic or bisphosphonates beads.

AFM gives morphological and lattice data to the nanometer scale. It also provides hardness information and

allows one to count the crystals on a grid.

SEM gives 3D images on a nanometer scale and can provide crystal composition. FITR can distinguish

between BCP and CPPD, but it is also destructive.

Raman spectroscopy is a better tool. The Raman spectrum of each type of calcium-containing crystal is

unique, allowing crystal characterisation. It is non-destructive and can distinguish between BCP and CPPD

crystals.

In conclusion, the development of simple, bedside diagnostic test for BCP crystals would enhance the

knowledge in their pathogenic effects.

Globally Optimizing Ultrasound Protocol, Standards, and Criteria for CPPD and Gout Walter Grassi, MD Clinica Reumatologica Scuola di Specializzazione in Reumatologia

Università Politecnica delle Marche

The use of ultrasound in diagnosing gout has been ignored for a long time but there are still a lot of barriers to

its use, for example the huge variability of findings from double lines to spots within the cartilage.

In sonography gout can have various manifestations:

1. Hyperechoic punctiform spots

2. Foggy synovial fluid

3. Dense microdotted fluid (uratic sand)

4. Polymorphic echogenic aggregates of MSU crystals without acoustic shadows (soft tophus)

5. Homogeneous MSU crystal aggregates generating acoustic shadow (hard tophus)

6. Intra-osseous tophaceous aggregates.

7. Intratendinous MSU clouds and/or cystal aggregates with or without acoustic shadow

8. Peritendinous/subcutaneous MSU aggregates

The pattern of recognition is a sort of Morse code of dots and lines, the dots indicating increased sensitivity

and decreased specificity and the lines decreased sensitivity and increased specificity. There are similar

morphological characteristics between gout and Calcium pyrophosphate deposition disease (CPDD).

In Gout, tendons and bursal involvement are indicated by dots clouds and stones. The clouds of UA crystal

aggregates interrupt fiber patterns of tendons.

The sonographic spectrum of CPPD crystal deposition disease is:

1. Polymorphic hyperechoic spots/clouds inside the hyaline cartilage

2. Hyperechoic spots around and inside fibrocartilage with or without acoustic shadow

3. Intra-articular hyperechoic punctiform spots

4. Intra-articular pyrophosphate tophi

5. Hyperechoic crystal aggregates at the condro-synovial interface

6. Intra-tendinous CPPD crystal aggregates with or without acoustic shadow

In CPDD the crystals are different. There is a wide variability of features. In CPDD there are double lines and

triple lines shown in the outer part of the cartilage. There are also dots and stones as in gout but they are

different in appearance.

To globally optimize ultrasound protocol, standards, and criteria for recognizing CPPD the gout research

agenda should consider making ultrasound the gold standard imaging technique for early diagnosis of gout

and CPPD. Also the cutoff level for diagnosis of crystal aggregate should be defined and an ultrasound

definition of MSU and CPPD tophi determined.

More research on pattern recognition and qualitative analysis in gout and CPPD is welcome because of the

wide spectrum of ultrasound findings.

The first metatarsophalangeal joint should be systematically assessed in patients with gout even if they are

asymptomatic. Additionally there should be systematic assessment of the patellar and Achilles tendon in

patients with suspected gout or CPPD and of the meniscus fibrocartilage, the triangular fibrocartilage complex

and the hyaline cartilage of the femoral condyles in patients with suspected CPPD.

Developing DECT Further for Assessment of Urate Burden Savvas Nicolaou, MD University of British Columbia

Dual Energy CT (DECT) uses two different X-ray sources and 2 data acquisition systems mounted on the

same X-ray gantry. Each X-ray source has an independent high voltage generator which allows for

independent control of both potential and current. Simultaneous low and high energy images can be

reconstructed with comparable image noise levels.

DECT has improved the ability to characterize and differentiate various body substances by material

decomposition. Differentiation is based on the fact that substances with higher molecular weight show different

attenuation behaviors at different energy levels. Attenuation measurements at tube voltages of 80 and 140 kV

can be subjected to 3-material decomposition, allowing for mathematical subtraction of substances.

Second generation DECT scanners allow the use of tin filters in the high-energy beam, performed by using 140

kV and additional filtration on the B tube and 80 kV on the A tube.

Beam hardening artifacts are found in DECT, the most commonly encountered being in the nails/nail beds of

the feet. In one study, this was seen in the majority of cases. It was much less common in the hand however.

This may be due to a similarity in Dual Energy Index (the value calculated from attenuation change with energy

level) between the keratinous nail bed and the monosodium urate.

One of the current issues with using DECT is that there is no real validated standardized grading system.

Results can vary with technique, different CT scanners, and urate burden score calculation. A consensus

paper to define the standard is required.

Another issue is reassessment of the tophus burden after treatment: Should we treat to target? In

asymptomatic hyperuricemia, is a screening imaging test required and what is its clinical utility?

It is also unknown whether interventions to reduce serum urate concentrations in such individuals with

hyperuricemia and imaging features of MSU crystal deposition have a role in reducing the risk of developing

recurrent flares or joint damage associated with gout.

A recent study currently in press found that in patients with asymptomatic hyperuricemia, the prevalence of

MSU deposition assessed by DECT was low; however, larger studies need to be performed for further

validation of this.

Additionally, visceral urate deposits are found in other organs. Urate burden in a CVS prostate study found

them in heart valves and prostate and may indicate a possible link to prostatitis.

DECT can be used in assessment of urate volumes in gout. DECT’s real power is that it is a classification tool.

A study to determine the specificity and sensitivity of DECT for gout; and the interobserver and intraobserver

reproducibility for DECT urate volume measurements was conducted on 40 crystal-proven gout patients (17

tophaceous) and 40 controls with other arthritic conditions.

The study concluded that DECT tophus volume measures are highly reproducible and that specificity of DECT

scans for gout was high, but sensitivity was more moderate (this may be due to use of urate lowering

therapies).

This study supports the promising clinical utility of DECT to accurately confirm the presence of MSU deposits

and the high reproducibility in assessing urate crystal volumes.

Other advantages of DECT:

DECT can be used in assessment of urate volumes in gout before and after treatment.

You can color code collagen as well caused identifying tendon inflammation and see if deposition is

due to Urate deposition.

DECT can be useful in tophus assessment. For example, a patient who presented with acute pain in

both ankles had an MRI. The MRI was non-diagnostic but a DECT showed intraosseous gout lesions.

DECT has been used in calcium scoring as an effective screening tool in low to moderate risk patients for

cardiovascular disease. There may be parallels in using DECT to score the total urate burden. Scores for urate

deposition are being developed. We can do topograms in areas of deposition and get a total burden of urate

throughout the body.

Recently, a newer application of DECT has provided an innovative way to image gout; DECT-Cinematic

Rendering, which allow for much more realistic 3D images.

What is in the future for DECT?

Develop urate score similar to calcium

• Need consensus paper with a validated standardized template for reporting and a grading scheme

– Quantify total urate burden - CT Scanogram, Cinematic Rendering

– Describe image quality, Standardize CT Technique and urate quantification among different

vendors

Session on G-CAN Lead Project 4

How Do We Narrow Disparities in Health Care Access Education and Quality of

Outcomes in Gout?

Gout in the Underserved; Prevalence, Disparity Jasvinder Singh, MD MPH University of Alabama at Birmingham

Physician-diagnosed prevalent gout is found in 3.9% of the total population in the United States; 4.0% (95% CI,

3.3%–4.8%) in Caucasians; 5.0% (95% CI, 3.3%–6.6%) in blacks.

In the U.S. racial minorities and those in a lower socio-economic status are not receiving the care needed to

manage their disease. They also have poorer outcomes.

When the outcomes are compared between racial minorities and Caucasians with gout, the minorities have a

higher baseline pre-treatment serum urate, higher use rates of emergency health care services, and a lower

rate of adherence to urate-lowering therapy. Minorities are also less likely to receive any urate-lowering

therapies such as Allopurinol and subsequently have a lower rate of success in lowering serum urate.

There are a number of factors that contribute to worse outcomes in racial minorities with gout, including:

• Socio-economic disparity

• Education

• Health care access

• Lower health literacy and numeracy

• Poorer physician-patient communication

• Higher medication non-adherence

• Greater risk averseness to treatments

Gout is like other chronic symptoms that require medical management. But for minorities, barriers exist that

prevent them from getting proper care. These barriers need to be understood and addressed for optimal

treatment. There are certain characteristics unique to this population that need to be considered:

◦ Education – not realizing that ULT is the key treatment or that diet management is integral to the

treatment of gout

◦ Concerns about medication adverse effects/interactions, cost

◦ Doubts about effectiveness of ULT

◦ Concomitant medications

◦ Forgetfulness, refilling the prescriptions on time

◦ Pill size and difficulty in swallowing

◦ Patient preference for alternative medicines

◦ Frequent travel; competing priorities.

We conducted a study with nine nominal groups of African-Americans with gout. The groups participated in

sessions lasting 1 to 1.5 hours each where they were given the opportunity to present and discuss their

concerns about treatment. Concerns were combined and rank-ordered. Quotes from these sessions

demonstrate many of the barriers that need to be overcome if we are to reduce disparities in treatment and

outcomes:

“I will forget because I am taking so many other medications.”

“I just got tired of being sick on my stomach.”

“Sometimes I am out of town, and did not have my medication with me.”

“I don’t like to take medicine.”

“Copay was $20- I have to ration the pills; I go with the pill I need the most, when I do that.”

Breaking these barriers to care and improving ULT adherence requires a systematic approach. The proposed

approach would include three interventions:

Patient-Centered Interventions:

◦ Focus on education about pharmacologic therapy

◦ Encourage lifestyle modifications with medication and diet, self-efficacy, coping skills

System-Based Interventions:

◦ Improvement of pharmacy process barriers

◦ Implementation of multidisciplinary and multifocal education programs

◦ Adequate support of medication management strategies

Provider-Based Interventions:

◦ Communicating risks and benefits of therapies

◦ Discussing coverage options

◦ Asking about barriers to adherence

We also propose a 12-month study to examine and eliminate barriers to serum urate testing in the

underserved. This should be a randomized, multicenter, two-arm study that looks at usual care versus systems

and technology-based interventions, including patient and physician education with and without EMR-based

interventions. Follow-up serum urate testing would be done with the primary outcome being to look at the

proportion of participants with a target serum uric acid of less than 6 mg/dl.

The secondary outcome would be urate-lowering therapy as measured by ULT adherence monitored through

medication refills. However it is important to include a component of patient satisfaction, HAQ and GAQ

scores, functional limitation and quality of life. The study should also look at health care utilization of ER/urgent

care and health care costs.

We propose a sample size of 200 to 300. The collaborators would be with any who has underserved

populations (Blacks, Hispanics, Lower SES) – and like trials!

It is important to target the underserved because of the potential for improved outcomes – you “get more for

your buck” if you focus on minority patients.

Insight and Models of Gout Care in the Underserved in New Zealand Lisa Stamp, MD, PhD Canterbury District Heath Board

Problem:

The Māori and Pacific (Polynesian) population of New Zealand (NZ) has the highest prevalence of gout

worldwide. However, the two primary challenges in managing gout in these groups are that people aren’t

presenting for healthcare and that rural areas of New Zealand are underserved by primary and tertiary care.

Most gout is managed in primary care settings in rural areas. However the guidelines are aimed at secondary

and tertiary health care providers. So how do we communicate best practice to our colleagues in primary care

to reach these underserved groups?

An interactive site map of New Zealand shows that rheumatologists are not in the areas where the need is

greatest. There are areas of high deprivation. The question becomes how do you get good healthcare to those

people in rural areas and engage them?

Solutions:

Primary care access

There is a need to think about simplified best practice protocols for urate lowering treatments, target UA and

comorbidities. Approaches to reach the underserved areas should include Telemedicine and online resources

such as health pathways and GP software for gout. Also nurse and pharmacist-led clinics can be effective in

areas where there are fewer physicians and rheumatologists. They can monitor the patients as well as refer

them directly to secondary care.

Engagement

There is a need for appropriate patient educational material. This should include standardized materials that

are targeted to the audience: Patient education is not “one-size-fits-all.” Pharmacists can also help engage

patients through SUA monitoring for those with gout on ULT as well as refer them directly to secondary care.

Session on G-CAN Lead Project 4

Advancing the Translation State of the Art for Joint Inflammation in Gout

Development of Gout Anti-Inflammatory Therapy.

Gauging the value and potential impact of emerging complementary therapy options

Cherry Products and Omega-3 Fatty Acids in Internet-Based Studies Tuhina Neogi, MD, PhD, FRCPC Associate Professor of Medicine and of Epidemiology

Boston University Schools of Medicine and of Public Health

Patients are using and will continue to use complementary therapies, so we need to address this. Cherries and

omega-3 fatty acids are an example of complimentary therapies thought to have some effect on gout.

There have been some limited studies linking cherries and a lower urate: in vitro inhibition ion in the presence

of cherries and reduction of flares in a small human study.

• Lower SUA: uricosuric +/- XOI activity

• Anti-inflammatory, antioxidant properties (anthocyanins)

• In vitro monocyte IL-1β, TNF-α release inhibited in response to MSU

• Possible reduction in flares

• Additionally there is evidence that Omega 3 polysaturated fatty acid decreased joint counts in RA

• Omega-3 polyunsaturated fatty acids have shown:

• Suppression of NLRP3 inflammasome in vitro and IL-1β in vivo

• Anti-inflammatory effects in RA

There are many different types of fatty acids. Omega 6 are pro-inflammatory, so the omega-3/omega 6 ratio is

important.

A Boston online gout case-crossover study looked at subjects with physician-diagnosed gout. When a subject

has a gout attack they log on and finish a questionnaire about their gout attacks and the 48 hours prior. The

time prior to an attack is the hazard period and the time when there was no gout attack is the control period.

The risk factor is assessed during the hazard and control periods with the same person i.e. each person acts

as their own control. Subjects recorded cherry or cherry extract intake in the prior two days. This was adjusted

for purine intake and use of alcohol, diuretics, Allopurinol, colchicine and NASAIDs. The study found that with

increasing consumption there was a decrease in risk of gout affects.

A similar study was done for omega-3 fatty acids. Studies were also done looking at supplements but they

could not account for dosing. Results were null.

There is sufficient observational data to test cherry and omega-3 supplementation as putative adjunctive

therapies and develop evidence recommendations for them.

Our proposal is to develop evidence-based recommendations for non-pharmacologic approaches to gout

management. There is a lack of evidence-based guidance regarding specific “dosing” recommendations.

Randomized, controlled trials are needed with dosing ranging studies. Comparators are needed to identify

appropriate comparator interventions that don’t make it worse e.g. the effect of low-dose fish oil vs. high-dose

sunflower oil as supplements are often mixed with the sunflower oil.

G-CAN could develop a gout clinical trials consortium to address the general need for clinical studies. This

should be overseen by specialists with experience in gout RCTs and include a network of clinicians with

interest in gout and the ability to recruit. Ideally it would also include sites with imaging capabilities (US,

DECT).

A study of cherries and cherry extract would include ongoing collaboration with interested investigators.

Omega-3 supplements studies could include Abhishek Abhishek, Catherine Hill, and any others that would be

appropriate.

Omega-3 Fatty Acids for Preventing Acute Attacks of Gout: Assessment by RCT

Abhishek Abhishek, PhD Clinical Associate Professor of Rheumatology

University of Nottingham

Urate lowering treatments have side effects and risks associated with them. There is a need for safe &

effective prophylaxis against gout attacks.

Gouty inflammation is driven by NLRP3 inflammasome-mediated IL-1β. Omega-3 fatty acids are known to

suppress activation of the NLRP3 inflammasome in vitro in macrophages, and limit experimental IL-1beta

mediated inflammatory responses in vivo. Thus it makes sense to study its efficacy for treatment.

20 years ago a mouse study found that mice given fish oil had less inflammatory infiltrate. The findings in mice

were confirmed by a Boston study.

Since then, a number of studies have looked at whether treatment with omega-3 fatty acids prevents acute

attacks of gout.

Healthy volunteer studies and studies in rheumatoid arthritis found that taking 2.4 - 2.7 gm/day of omega-3

fatty acids reduces unstimulated IL-1β, IL- 6, CRP and TNF α concentration by up to 70% - 80% and

lipopolysaccharide stimulated TNF α and IL-1β production by over 70%, thus reducing gouty inflammation.

The anti-inflammatory effects of omega-3 fatty acids become apparent in 4 weeks. It improves the patient’s

response to methotrexate and reduces NSAID use in RA as well. Higher doses do not have any additional anti-

inflammatory effect. More than 4gm a day has no additional benefits.

Since it takes up to four weeks for anti-inflammatory effects to appear it is not effective as a treatment but as a

prophylactic.

We propose the following studies.

Proposal I. A pilot proof of concept randomized trial.

Eligible cases will be recruited for ULT as per British guidelines from primary care.

Subjects will be given 4 gm/ day of omega-3 fatty acid in soft-gel capsules. These will be given for only the first

four weeks beginning at a low dose. The placebo will be olive oil filled capsules. It will give us the idea of effect

size and allow us to calculate sample size for a trial. At 28 weeks observational and mechanistic studies will be

done. This will allow us to look at the appropriate dose.

Proposal 2. A large definitive RCT and a minimum effective dose finding RCT

In the definitive RCT subjects will be given 4 gm/ day of omega-3 fatty acid in soft-gel capsules. These will be

given for only the first four weeks beginning at a low dose. The placebo will be olive oil filled capsules.

For the minimum effective dose finding study, subjects will be given either 2, 3 or 4 gm/ day of omega-3 fatty

acid in soft-gel capsules.

New frontiers for small molecule inhibitors of gouty inflammation

Resolution Mechanisms and Coupling with Tophus Formation Martin Herrmann, MD University Erlangen

Gout is a prototypical self-resolving inflammation. Acute gout attacks are the result of activation of the

inflammasome which occurs when the MSU crystals are ingested by neutrophils and monocytes. So why

doesn’t the inflammation continue indefinitely?

Once the neutrophils ingest MSU crystals they undergo a form of cell death characterized by the formation of

neutrophil extracellular traps (NETs). A mouse study found that an oxidative burst is required to prevent MSU-

induced bone destruction. At 46 days if you block NET, the anti-inflammatory effects of the NET are gone. We

can conclude from this that reactive oxygen species (ROS) are required to resolve neutrophil-driven

inflammation in gout, which makes neutrophil-derived-ROS “good guys.”

The tophi seen in gout patients are composed of NETs and densely-packed MSU crystals. At high neutrophil

densities, NETs tend to aggregate in vitro. You can see them develop in the plate. But if you look for

supernatant there are no cytokines, which are usually present in inflammation. Materials are able to degrade

cytokines before they are released and the tophus degrades.

Proposal I

I propose to investigate tophi from human surgical material for neutrophil signature and for signs of

inflammation. I also I propose to extend the study to MSU-kidney stones and to blood infected with malaria.

Proposal 2

I am looking for co-operation with surgeons who have access to tophaceous material from humans. Nicola

Dalbeth is a candidate. We can offer murine models for drug testing.

Markers of Tophus Formation and Inflammatory Connective Tissue Destruction Nicola Dalbeth University of Auckland

A tophus is an organized response to MSU crystals. Innate and adaptive immune cells are present in the

tophus. They are seen in gout patients with severe advanced disease and often long-standing, poorly treated

gout ulcerating infections. The consequences to patients are poor function of hands and feet and footwear

problems.

Tophi are closely related to structural joint damage and CT and MRI scans show bone erosion and the

presence of MSU crystals. The mechanism for this is reduced osteoblast mediated bone formation and

increased osteoclast bone reabsorption leading to localized bone erosion. There are similar mechanisms in

cartilage damage involving increased degradative enzymes and metabolic pathways. Tophi result in active

damage to cartilage and possible passive destruction of tendons.

The key questions are:

• Why do tophi form in some people with hyperuricaemia and not others?

• Can we predict which patients with gout will develop tophi and joint damage?

• What are the key mechanisms of structural joint damage in gout?

• What is the best strategy for treating patients with joint damage?

The approach to answer these questions is a cohort study using clinical, imaging and lab evidence including a

hand function study and foot function study with XR and CT evidence. Additionally there should be a

longitudinal observational study of patients who have had gout of less than ten years duration supported by

serial XR and a Zolgout RCT with serial XR and CT, including DECT.

New Allopurinol randomized controlled trials with very comprehensive imaging using serial XR and DECT are

to be reported in the next year. Funding has also been obtained for a new RCT of intensive ULT for

tophaceous gout using serial XR and DECT.

We have developed several new research tools including a questionnaire for patient-reported outcome

measure of tophus burden available at www.fmhs.auckland.ac.nz/TIQ-20 and a novel semi-quantitative DECT

scoring method for measurement of urate deposition in the feet and ankles which are is also available to use.

The above articles represent a synopsis of the presentations made at the G-CAN 2015

Inaugural Scientific Meeting November 6, 2015 in San Francisco, California USA.

You are invited to submit your ideas for G-CAN projects to

hyonchoiandbobterkeltaub@g-can.org

The 2016 G-CAN Annual Meeting is scheduled for Friday, November 11, 2016 at the Westin DC City Center in Washington,

DC, USA. Register online after July 1 at www.123Signup.com/G-CANMEETING. Thank you!