Population-specific association between ABCG2 variants and tophaceous disease in people with gout

The gouty tophus consists of monosodium urate (MSU) crystal deposits surrounded by chronic granulomatous inflammatory tissue [1]. In people with gout, tophi contribute to musculoskeletal disability, joint damage and poor health-related quality of life, and are associated with increased risk of mortality [2‐5]. Typically, tophi present as subcutaneous nodules many years after initial presentation with acute inflammatory flares [6]. Although advanced age, kidney disease and diuretic use have been reported to be risk factors for development of tophi (reviewed in [7]), few studies have systematically examined the features associated with tophaceous disease.

In the last decade, there has been major progress in understanding the genetic basis of hyperuricaemia and gout. Variants in two genes, ABCG2 and SLC2A9, are consistently associated with hyperuricaemia and prevalent gout in many different populations [8‐10].

Aotearoa New Zealand has a high prevalence of gout, with early onset, severe disease in Māori and Pacific people [11, 12]. The SLC2A9 rs11942223 risk allele is strongly associated with prevalent gout in Māori and Pacific people living in Aotearoa New Zealand [13]. In contrast, the ABCG2 rs2231142 risk allele (Q141K) has population-specific effects in Polynesian people, with a strong association with gout in people of Western Polynesian (Tonga, Samoa, Niue and Tokelau) ancestry, but a weak effect in people of Eastern Polynesian (New Zealand Māori and Cook Island Māori) ancestry [14].

Although the association with gout is well-established in many populations, it is unclear whether variants in these genes also contribute to phenotypic differences in people with gout. We have previously reported that a non-synonymous SLC2A9 Arg265His variant is associated with tophi in New Zealand Māori with gout [15], and a Taiwanese study has reported an association between ABCG2 Q141K and tophi in both Han Chinese and Taiwanese aboriginal people with gout [16]. The aim of this study was to examine the role of SLC2A9 and ABCG2 variants in tophaceous disease in people with gout.

People with gout were recruited into the study, Genetics of Gout in Aotearoa, from primary and secondary care in Aotearoa New Zealand (n = 1778) [10]. The study was designed to identify genetic and clinical factors associated with gout. All participants in this analysis fulfilled the 1977 American Rheumatism Association (ARA) preliminary gout classification criteria [17]. The New Zealand Multi-Region Ethics Committee granted ethical approval (MEC/05/10/130) and all patients provided written informed consent.

All participants attended a study visit, which included a detailed clinical assessment. This included recording of age of onset of the first presentation with gout, flare frequency in the previous year (self-reported), medications, physical examination of weight and height, and serum creatinine testing. The presence of palpable tophi was assessed by experienced research assistants with training in the clinical assessment of gout. The highest recorded serum urate concentration was obtained from community electronic laboratory records. Presence of comorbid conditions (specifically cardiac disease, hypertension and type 2 diabetes mellitus) was recorded and verified using the electronic medical record. The estimated glomerular filtration rate (eGFR) was calculated using the modification of diet in renal disease formula [18].

SLC2A9 rs11942223, ABCG2 rs2231142 and ABCG2 rs10011796 single nucleotide polymorphisms (SNPs) were genotyped using TaqMan SNP genotyping assay technology (Applied Biosystems) [10]. SLC2A9 rs11942223 and ABCG2 rs2231142 were selected as variants consistently associated with gout in many populations, including Polynesian populations (reviewed in [9]). ABCG2 rs10011796 was selected as an additional gout-associated ABCG2 SNP [8], noting that this SNP has also been associated with allopurinol response in a USA study population [19]. The linkage disequilibrium (r ²) was <0.11 between the two ABCG2 SNPs for all ancestral groups. Risk allele positivity was analysed for the ABCG2 SNPs. However, owing to the high prevalence of the risk allele at SLC2A9, particularly in Polynesian people, minor allele (T) positivity was analysed instead.

Data were analysed in SPSS v22 (SPSS Inc., Chicago, IL, USA). Clinical and genetic features were analysed according to tophus status (tophi vs no tophi). Pearson’s chi-squared test (categorical variables) and the two-sample t test (continuous variables) were used to compare the two groups. P < 0.05 was considered significant for this analysis and was not corrected for multiple testing due to prior evidence of genetic effects in gout and tophus. For the detailed ABCG2 analysis, logistic regression was performed on the presence/absence of the risk allele with tophus status, adjusted by potential confounders. Polynesian ancestry was separated into Western Polynesian (Tonga, Samoa, Niue or Tokelau) and Eastern Polynesian (Māori or Cook Island Māori) [14]. In the case of mixed Polynesian ancestry with Māori (n = 29), patients were analysed as Māori (Eastern Polynesian) as recommended by the New Zealand Ministry of Health.

This study provides evidence that variation in ABCG2 function may play a role in the development of tophaceous disease in some populations with high prevalence of severe gout. This effect appears to be independent of other risk factors such as serum urate concentrations or disease duration.

High prevalence of gout has been reported in indigenous Māori and in Pacific people residing in Aotearoa New Zealand, with contemporary rates of 11.7% in Māori men and 13.5% in Pacific men, compared to 3.7% in European men [11]. Our study has shown higher prevalence of tophaceous disease in people with gout of Māori or Pacific ancestry. Consistent with prior studies [6, 20, 21], we have also demonstrated that disease duration is associated with tophaceous disease in people with gout. Although serum urate concentrations were associated with tophaceous disease in participants of non-Polynesian ancestry, this association was not observed in the participants of Māori or Pacific ancestry. This study has also highlighted the impact of disease in patients with tophi, with more frequent flares and greater use of anti-inflammatory therapy, compared to people with gout without tophi.

The key finding of this study is the ancestry-specific differences in the association between ABCG2 variants and tophaceous disease in people with gout. In particular, the associations with the ABCG2 SNPs were observed consistently only in the Western Polynesian ancestral group. Association of a similar effect size between rs2231142 (Q141K) and tophi has been reported in both Han and aboriginal Taiwanese people with gout (OR 1.51 and 1.50, respectively, for the 141 K allele) [16]. Other data also support the ancestry-specific effects of ABCG2 in gout; we have previously reported that compared to people without gout, the rs2231142 risk allele (141 K) is associated with susceptibility to gout in New Zealanders of European and Western Polynesian ancestry, but not in those of Eastern Polynesian ancestry [14]. Importantly, the frequency of the rs2231142 risk allele is substantially higher in people of Western Polynesian ancestry, compared with the other ancestral groups: in a prior case-control study comparing people with gout with control participants with gout [14], the minor allele frequency in people unaffected by gout was 27.5% in Western Polynesians, 9.0% in Eastern Polynesians and 12.6% in Europeans; the minor allele frequency in people with gout was 51.9% in Western Polynesians, 10.7% in Eastern Polynesians and 24.2% in Europeans.

We also report a novel association between another ABCG2 SNP rs10011796 and tophi in people with gout who are of Western Polynesian ancestry. This ancestry-specific association between tophi and rs10011796 was independent of rs2231142 (Q141K). Together with rs2231142, this SNP has been associated with allopurinol response in a USA study population of people with gout [19]. Of note, the strong effect of rs10011796 on allopurinol response in the USA study was attenuated after adjusting for ancestry, further suggesting ancestry-specific effects [19]. The ancestry-specific effects at ABCG2 may relate to different haplotypic backgrounds between the Western Polynesian, European and Eastern Polynesian populations. This possibility could be evaluated by specific resequencing of this locus in people of Polynesian ancestry.

The mechanisms of association between ABCG2 and tophaceous disease in people with gout are currently unclear. Both SLC2A9 and ABCG2 are associated with hyperuricaemia in the general population, and the lack of an observed association between tophaceous disease and SLC2A9 rs11942223 suggests that the association between ABCG2 and tophaceous disease in people with gout are not entirely due to effects on serum urate concentrations. This conclusion is further supported by the persistent associations with ABCG2 after adjusting for serum urate and disease duration. The ABCG2 risk alleles have been associated with allopurinol resistance, and it is possible that these variants lead to relative hyperuricaemia even with urate-lowering therapy, increasing the risk of tophaceous disease [19, 22]. ABCG2 (also known as breast cancer resistance protein (BCRP)) is ubiquitously expressed (reviewed in [23]), and it is also possible that altered ABCG2 function may regulate other factors contributing to tophus formation, such as promotion of crystal formation or the inflammatory response to deposited crystals.

In our study, the association between ABCG2 variants and tophaceous disease persisted in people with gout of Western Polynesian ancestry even after adjusting for potential confounders such as highest recorded urate, disease duration and serum creatinine. These findings raise the possibility that the relationship between ABCG2 and tophaceous disease are independent of the effects of prolonged hyperuricaemia.