**1. Introduction**

Serum urate concentration is a complex phenotype influenced by both genetic and environmental factors, as well as interactions between them. Hyperuricemia results from an imbalance between endogenous production and excretion of urate. This disorder is a central feature in the pathogenesis of gout [1], which progresses through several degrees, i.e., asymptomatic hyperuricemia, acute gouty arthritis, intercritical gout, and chronic tophaceous gout. While not all individuals with hyperuricemia develop symptomatic gout, the risk of gout increases in proportion to the elevation of urate in circulation. In addition to hyperuricemia, the risk is also associated with gender, weight, age, environmental,

**Citation:** Toyoda, Y.; Pavelcová, K.; Bohatá, J.; Ješina, P.; Kubota, Y.; Suzuki, H.; Takada, T.; Stiburkova, B. Identification of Two Dysfunctional Variants in the ABCG2 Urate Transporter Associated with Pediatric-Onset of Familial Hyperuricemia and Early-Onset Gout. *Int. J. Mol. Sci.* **2021**, *22*, 1935. https://doi.org/10.3390/ijms22041935

Academic Editor: Thomas Falguières Received: 30 November 2020 Accepted: 10 February 2021 Published: 16 February 2021

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and genetic factors [2,3], and interactions between them all. Recent data suggest that the number of gout patients under the age of 40 years (early-onset) is increasing [4]. These early-onset patients may have different clinical signs and co-morbidities from those who present with gout at a later age [5,6]. Given the development of earlier metabolic disorders in the early-onset gout patients compared with common gout patients [5], together with the need for continuous management of health from their younger age, understanding the risks of early-onset gout is clinically important.

More and more evidence suggests that the net amount of excreted uric acid is mainly regulated by physiologically important urate transporters, such as urate transporter 1 (URAT1, known as SLC22A12, a renal urate re-absorber) [7], glucose transporter member 9 (GLUT9, also known as SLC2A9) [8,9], and ATP-binding cassette transporter G2 (ABCG2, a high capacity urate exporter expressed in the kidneys and intestines) [10–13]. Dysfunction of URAT1 and GLUT9 reportedly cause inherited hypouricemia type 1 and type 2, respectively, while dysfunction of ABCG2 is a risk factor for hyperuricemia and gout [1,14]. Additionally, the ABCG2 population-attributable percent risk for hyperuricemia has been reported to be 29.2%, which is much higher than for those with more typical environmental risks such as BMI ≥ 25.0 (18.7%), heavy drinking (15.4%), and age ≥ 60 years old (5.74%) [15]. Hence, dysfunctional variants of ABCG2 may affect clinical outcomes by influencing the accumulation of uric acid in the body.

The ABCG2 protein, which is an *N*-linked glycoprotein composed of 655-amino acid, is a homodimer membrane transporter found in a variety of tissues [16–18]. ABCG2 is expressed on the brush border membranes of renal and intestinal epithelial cells, where ABCG2 is involved in the ATP-dependent excretion of numerous substrates from the cytosol into the extracellular space. ABCG2 was historically first described as a drug transporter linked to breast cancer resistance [19–21], which led to many studies that focused on its critical role in drug pharmacokinetics. To date, not only xenobiotics but also endogenous substances, including uremic toxin [22] and urate [11,12], have been identified as ABCG2 substrates.

In the context of hyperuricemia/gout, there are about 50 allelic variants, including a number of rare variants with minor allele frequencies (MAF) < 0.01%, which have been found in the *ABCG2* gene. Wide ethnic differences have been found relative to the frequencies of these alleles. There are two well-studied, common ABCG2 allelic variants p.V12M (c.34G>A, rs2231137) and p.Q141K (c.421C>A, rs2231142) that have highly variable frequencies depending on ethnicity. Both are commonly found in Asians (in a relatively large number of ethnic groups) but are rarely found in Europeans [23]. A minor allele of p.V12M appears to be protective regarding susceptibility to gout [24]; however, this apparent effect is due to linkage disequilibrium between p.V12M and other dysfunctional ABCG2 variants [25]. In other words, the V12M mutation has little impact on the function of ABCG2. On the other hand, the p.Q141K variant decreases ABCG2 levels, which reduces the cellular function of ABCG2, as a urate exporter, by 50% [12]. In addition to p.Q141K, p.Q126X (c.376C>T, rs72552713), which is common in the Japanese population (MAF, 2.8%) [26] but rare in other populations, has been identified as hyperuricemia- and gout-risk allele [12]. Given that p.Q141K and p.Q126X variants are both associated with a significantly increased risk of gout, the effects of dysfunctional variants of ABCG2, relative to gout susceptibility, are genetically strong [27].

In a recent study focusing on 10 single nucleotide polymorphisms in 10 genes (*ABCG2*, *GLUT9*/*SLC2A9*, *SLC17A1*, *SLC16A9*, *GCKR*, *SLC22A11*, *INHBC*, *RREB1*, *PDZK1*, and *NRXN2*) that are strongly associated with serum uric acid (SUA) concentrations, only ABCG2 p.Q141K was associated with early-onset gout (< 40 years of age) in European and Polynesian subjects [28]. Additionally, in a previous study, we found that the MAF of p.Q141K in a cohort of hyperuricemia and gout with pediatric-onset was 38.7%, which was significantly higher than adult-onset (21.2%) as well as normouricemic controls (8.5%) [29]. This information suggests that ABCG2 dysfunction could be strongly associated with pediatric-onset hyperuricemia and early-onset gout; however, compared with adult-onset

gout, little clinical information is available, except for the ABCG2 p.Q141K, regarding earlyonset gout linked to other SUA-associated mutations. Furthermore, the latent involvement of ABCG2 in the development of this disease requires further evidence.

In this study, we investigated the genetic cause of pediatric-onset hyperuricemia and early-onset gout over three generations of a single family. Based on a positive family history of hyperuricemia or gout, we identified two rare mutations, c.393G>T (p.M131I) and c.706C>T (p.R236X), in the *ABCG2* gene. A series of biochemical assays revealed that ABCG2 p.M131I and p.R236X were functionally deficient and null, respectively. Our results also contributed to a more in-depth understanding of the effects of rare *ABCG2* variants, which is a highly polymorphic gene, on its function as a physiologically important transporter.
