**5. ABCG2 Polymorphisms in Pediatric-Onset Hyperuricemia and Early-Onset Gout**

Hyperuricemia and gout pathology has often been shown to be related to genetic predisposition [30] and to be affected by SNPs in many of the genes encoding urate transporters [87,88]. Among these, especially SNPs of ABCG2 have been highly associated with pediatric-onset hyperuricemia and early-onset gout [89–93]. These polymorphisms are summarized and organized in Table 1 according to the standard nomenclature rules for molecular diagnostics [94]. Furthermore, the localization of these polymorphisms on the protein sequence of ABCG2 is shown in Figure 1. It should be noted that there is a subset of other function-impairing SNPs in ABCG2 [95,96], but most of them have not yet been associated with pediatric hyperuricemia or early-onset gout. One of the best-studied variations of the ABCG2 amino acid sequence is the previously discussed Q141K polymorphism, which also gives rise to other important clinical phenotypes, such as in the pharmacokinetics and tissue distribution of drugs transported by ABCG2 [97]. The polymorphism is highly associated with early-onset hyperuricemia, gout, and hyperuricemia-associated comorbidities, which cause a high mortality rate in hemodialysis patients [98]. Although the F489L polymorphism has not been as well studied in the context of disease, it shows a similar inhibitory effect on the ABCG2 transport function as the Q141K mutation. As with the Q141K mutation, ABCG2 carriers with the F489L mutation show reduced expression and reduced ABCG2 transport capacity [75]. Inhibition of proteasomal degradation could partially restore the transport function of both ABCG2 variants. In contrast to the Q141K polymorphism, which causes amino acid sequence alterations in the nucleotide-binding domain, the F489L polymorphism is localized in the transmembrane domain. This shows that the impairment of ABCG2 function can be caused by changes in amino acid structure in different domains of the transporter. Polymorphisms in the transmembrane domain have often been associated with decreased surface expression of the ABCG2 transporter and impaired substrate transport abilities [99]. However, there is not much literature to support their clinical impact in both late and early-onset hyperuricemia and gout. The clinical importance of a certain polymorphism on the development of hyperuricemia and gout usually is related to its minor allele frequency in humans and its functional impact on the protein of interest. Due to genetic drift caused by spatial separation of populations, certain polymorphisms have accumulated in different ethnicities. For example, the frequency of V12M polymorphism is high in Mexican Indians but low in Caucasian and Middle Eastern populations [97]. In contrast, the Q141K and Q126X polymorphisms are enriched in Japanese populations, whereas in Caucasians, Q141K is not as common and Q126X is virtually absent [97]. Our understanding of the genetic variations in the ABCG2 sequence associated with hyperuricemia and gout is still incomplete, as evidenced by the recent discovery of less common polymorphisms previously unrecognized or not studied in the context of hyperuricemia and gout [89,90,95,100]. Two of these newly identified rare polymorphisms have been recently described in a case report of a 12-year-old Czech girl of Roma ethnicity with chronic asymptomatic pediatric-onset of hyperuricemia [89]. In this regard, several rare diseases have been found to occur primarily or exclusively in individuals of Roma ethnicity, and many of the mutations underlying these diseases have been recently discovered, such as for Charcot-Marie tooth disease types 4D and 4G [101,102], the congenital cataract facial dysmorphism neuropathy [103], the Gitelman syndrome [104], and the Galactokinase deficiency [105]. In the afore-mentioned case of the 12-year old girl, DNA sequencing analysis of the ABCG2 gene revealed the presence of heterozygously expressed missense (c.393G > T, p.M131I) and nonsense (c.706C > T, p.R236X) mutations (Figure 1, blue residues) causing the pediatric-onset of hyperuricemia observed in the girl's ancestry and the early-onset of gout especially in male individuals of the maternal line of inheritance. In the study, the functional consequences of the mutations were investigated

in comparative in vitro experiments. Due to the in-frame stop codon induced by the R236X mutation, the ABCG2 protein sequence was truncated to about 1/3 of the full-length protein, with the mutant protein lacking a functional transmembrane domain. Therefore, no plasma membrane localization and no urate transport activity of the mutant protein could be observed. In contrast, the M131I mutation was translated to a full-length protein with no impairments in N-glycosylation at residue N596 and normal membrane localization. However, the urate transport capabilities of the M131I mutant were reduced to <15% of wildtype levels [89]. M131 itself was found to be a highly conserved residue that is localized close to the Q-loop within the nucleotide-binding domain of ABCG2 (Figure 1). The conserved glutamine Q126 in the center of the Q-loop is responsible for the coordination of the magnesium ion associated with ATP in the catalytic center of the protein [68]. M131I may thus alter the spatial orientation of the Q-loop or sterically hinder the coordination of Mg-ATP, thereby drastically reducing ABCG2′ s ATP hydrolysis capabilities necessary for providing the energy for substrate transport across the membrane. Another newly identified polymorphism associated with pediatric hyperuricemia and early-onset gout is I242T, which was found in the lineage of another young European girl and was analyzed in a similar way [93]. Like the aforementioned M131I mutant, the I242T mutant ABCG2 variant showed no impairment in glycosylation and membrane localization, although its urate transport abilities were drastically reduced. This effect could be coincidentally related to the close localization of the mutants at the conserved H243 within the H-loop or also called histidine switch of the catalytic center of ABCG2 (Figure 1). The H-loop is responsible for coordinating the γ-phosphate of ATP, which is responsible for ATP hydrolysis. For further research, I242T and M131I may represent interesting new candidates to study the consequences of ABCG2 loss-of-function without disrupting ABCG2 membrane localization and protein-protein interactions. These representative case reports also show that depending on the severity of the disruption of the urate transportability of ABCG2, homoor heterozygosity of the dysfunctional polymorphisms and further genetic predispositions in other genes involved in urate homeostasis [106], hyperuricemia can already occur in childhood (pediatric-onset), which increases the risk for the development of early-onset gout. This allows the risk allele of a particular polymorphism to be identified and considered for clinical diagnosis. Interestingly, compared to patients with late-onset gout, patients with early-onset gout also show clinical symptoms that indicate a more severe disease pattern. This includes a prolonged disease duration, a different localization of the first occurring arthritis (with a lower incidence of typical metatarsophalangeal manifestations and a higher incidence of ankle- or mid-foot involvement in early-onset gout), a higher flare frequency (gout attacks), and an increased overall number of involved joints [15,16]. In terms of gout-associated comorbidities, late-onset gout patients are more likely to suffer from chronic kidney disease, metabolic syndrome, and cardiovascular disease, a phenomenon probably related to the age difference between the two patient groups [16]. However, these comorbidities occur at a younger age in patients with early-onset gout. In contrast, a recent study showed that patients diagnosed with gout at age 40 or younger may be at increased risk for cardiovascular disease and recurrent gout compared to those diagnosed later in life [107]. In this study, of 427 adult patients diagnosed with gout at a New England multispecialty group practice, 327 who were aged 40 years or younger at diagnosis were more likely to have cardiovascular risk factors. For example, these younger patients had a significantly higher body mass index than gout patients over 40 years of age, and a substantial proportion of the younger patients also suffered from hypertension or hyperlipidemia. Moreover, early-onset gout patients were less likely to achieve a serum uric acid level below 6.0 mg/dL after therapeutic intervention as compared to late-onset gout patients. Therefore, clinical screening for hyperuricemia in genetically predisposed families and prompt urate-lowering therapy in pediatric, adolescent, or young adult patients with still asymptomatic chronic hyperuricemia could help delay the onset of gout and the development of hyperuricemia-related comorbidities [108–110]. With regard to the treatment of cardiovascular comorbidities in hyperuricemia patients, it should be noted

that blood pressure-lowering drugs such as the AT1 receptor blocker telmisartan have been shown to inhibit the transport activity of ABCG2 [75] thereby potentially exacerbating hyperuricemia in patients with a corresponding genetic predisposition. In view of the emerging role of ABCG2 and its importance for intestinal excretion of uric acid, it may in principle represent a novel pharmacotherapeutic target to lower uric acid levels [43–47]. As speculation, this may be accomplished by modifying ABCG2 expression and function in intestinal epithelial cells. For example, in patients expressing mutant forms of ABCG2, this opens up the possibility of developing small molecule drugs with high pre-systemic elimination to target the function, cellular handling, or expression of ABCG2 predominantly in intestinal epithelial cells, thereby locally normalizing the impaired intestinal uric acid excretion in these individuals without interfering with the function of ABCG2 in other tissues (e.g., extrusion of xenobiotics). This area of research, therefore, shows great potential for the development of targeted pharmacotherapies for specific populations of genetically predisposed individuals with early-onset gout and thus warrants innovative research in the near future.

**Table 1.** Polymorphisms in ABCG2 protein sequence associated with pediatric-onset hyperuricemia and early-onset gout.

