**3. Discussion**

In this study, we found and analyzed a representative case of pediatric-onset hyperuricemia and early-onset gout in a Czech family associated with two newly identified, one functionally deficient and the other a null mutation, in *ABCG2*. A positive family history of hyperuricemia/gout in the context of ABCG2 dysfunction was observed in the maternal line (the mother, maternal uncle, and maternal grandfather) of the proband (a young girl, of Roma ethnicity, with chronic asymptomatic hyperuricemia); her father exhibited elevated serum urate that seemed to be associated with metabolic syndrome. Although a decrease in net renal urate excretion was observed in all cases of hyperuricemia in this family, which was characterized by FE-UA < 5%, our findings suggest that the hyperuricemia was linked to heterogeneity. Moreover, the familial hyperuricemia/gout observed in this study was not associated with a common variant, such as p.Q141K, but with a rare ABCG2 variant; this supports the recently proposed genetic concept, i.e., the "Common Disease, Multiple Common and Rare Variant" model [25,36], for the association between hyperuricemia/gout and the *ABCG2* gene.

Our findings of ABCG2 variants will provide deeper insights into amino acid positions that are critical for normal ABCG2 function. Based on cryo-electron microscopy (cryo-EM) of ABCG2 [37], both the M131 and R236 residues are in the cytoplasmic region of the N-terminus of the ABCG2 protein. Regarding p.M131I, the original amino acid (M131) sequence is conserved in several major mammalian species (Figure 3). Unlike the p.Q141K variant, which affects intracellular processing of the ABCG2 protein [38], the p.M131I variant disrupts ABCG2's function as a urate transporter, with little effect on the ABCG2 protein or its cellular localization. A plausible explanation for the molecular mechanism of the p.M131I effect is that this amino acid substitution could affect ABCG2 substrate specificity and/or affect its ATPase activity, including the binding affinity of ATP, which is the driving force for ABC transporters. The latter possibility is supported by a structural feature that the M131 residue is located near the conserved glutamine (Q126) within a Q-loop in the nucleotide-binding domain of ABCG2, a key element for the catalytic cycle of ATP binding and hydrolysis [37,39]. Further studies are needed to address this biochemical issue, which may deepen the mechanistic insight of ABCG2 protein. With regard to p.R236X, the acquired stop codon results in the production of a shortened ABCG2 variant (only about one-third the amino acid length of the native ABCG2 protein) that lacks all the transmembrane domains essential for normal protein function as a membrane transporter. This is consistent with our results demonstrating that the p.R236X variant is functionally null. Thus, the c.[393G>T; c.706C>T] (p.[M131I; R236X]) variant does not function as a urate transporter. *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 7 of 15 mechanism of the p.M131I effect is that this amino acid substitution could affect ABCG2 substrate specificity and/or affect its ATPase activity, including the binding affinity of ATP, which is the driving force for ABC transporters. The latter possibility is supported by a structural feature that the M131 residue is located near the conserved glutamine (Q126) within a Q-loop in the nucleotide-binding domain of ABCG2, a key element for the catalytic cycle of ATP binding and hydrolysis [37,39]. Further studies are needed to address this biochemical issue, which may deepen the mechanistic insight of ABCG2 protein. With regard to p.R236X, the acquired stop codon results in the production of a shortened ABCG2 variant (only about one-third the amino acid length of the native ABCG2 protein) that lacks all the transmembrane domains essential for normal protein function as a membrane transporter. This is consistent with our results demonstrating that the p.R236X variant is functionally null. Thus, the c.[393G>T; c.706C>T] (p.[M131I; R236X]) variant does not function as a urate transporter.


**Figure 3.** ABCG2 M131 is shown to be evolutionary conserved among seven mammalian species. Regarding the Abcg2 protein in each species, the NCBI Reference Sequence IDs are summarized as *Pan troglodytes* (Chimpanzee), GABE01009237.1; *Macaca mulatta* (Rhesus macaque), NM\_001032919.1; *Sus scrofa* (Pig), NM\_214010.1; *Bos taurus* (Bovine), NM\_001037478.3; *Rattus norvegicus* (Rat), NM\_181381.2; *Mus musculus* (Mouse), NM\_011920.3. Multiple sequence alignments and homology calculations were carried out using GENETYX software (GENETYX, Tokyo, Japan) and the ClustalW2.1 Windows program, per the protocol used in our previous study [40]. **Figure 3.** ABCG2 M131 is shown to be evolutionary conserved among seven mammalian species. Regarding the Abcg2 protein in each species, the NCBI Reference Sequence IDs are summarized as *Pan troglodytes* (Chimpanzee), GABE01009237.1; *Macaca mulatta* (Rhesus macaque), NM\_001032919.1; *Sus scrofa* (Pig), NM\_214010.1; *Bos taurus* (Bovine), NM\_001037478.3; *Rattus norvegicus* (Rat), NM\_181381.2; *Mus musculus* (Mouse), NM\_011920.3. Multiple sequence alignments and homology calculations were carried out using GENETYX software (GENETYX, Tokyo, Japan) and the ClustalW2.1 Windows program, per the protocol used in our previous study [40].

Czech family studied in this study is of Roma ethnicity. The Roma composes a transnational ethnic population of 8–10 million, with the original homeland being India; currently, they are the largest and the most widespread ethnic minority in Europe. The founder effect and subsequent genetic isolation of the Roma have led to a population specificity regarding the genetic background of specific human diseases. In other words, mutations associated with rare diseases found in the Roma population tend to be at extremely low frequencies in other European populations, and vice versa. Indeed, several genetic variants causing rare diseases are unique to the Roma, and many of these mutations have only recently been discovered, e.g., Charcot Marie Tooth disease type 4D and 4G (OMIM 601455 and 605285), Congenital cataract facial dysmorphism neuropathy (OMIM 604166), Gitelman syndrome (OMIM 263800), and Galactokinase deficiency (OMIM 230200) [41,42]. In light of the genetic specificity found in the Roma, we investigated the frequency of two identified *ABCG2* mutants (c.393G>T and c.706C>T) in our control cohort of 60 subjects of Roma origin (see Section 2.2 in Result*s*). MAFs of c.393G>T in the Roma cohort and European population were 0.833% (1 allele/120 subjects) and 0.002% (6 alleles/64,397 subjects), respectively. Although the sample size of our control cohort was very modest, it could be large enough to imply that the origin of c.393G>T might be the Roma population. On the other hand, the prevalence of c.706C>T was higher in Europeans than in the Roma, suggesting that these two *ABCG2* mutations could have different genetic origins.

For clinical practice, our findings suggest a need for further discussion about the potential benefits of urate-lowering therapy after a diagnosis of hyperuricemia in pediatriconset patients with ABCG2 dysfunction. Interestingly, harboring p.Q141K is reportedly associated with inadequate response to allopurinol (characterized by a smaller reduction

To discuss the expected independence in the genetic origin of two *ABCG2* mutations identified in this study, we will focus on the genetic specificity of each population. The Czech family studied in this study is of Roma ethnicity. The Roma composes a transnational ethnic population of 8–10 million, with the original homeland being India; currently, they are the largest and the most widespread ethnic minority in Europe. The founder effect and subsequent genetic isolation of the Roma have led to a population specificity regarding the genetic background of specific human diseases. In other words, mutations associated with rare diseases found in the Roma population tend to be at extremely low frequencies in other European populations, and vice versa. Indeed, several genetic variants causing rare diseases are unique to the Roma, and many of these mutations have only recently been discovered, e.g., Charcot Marie Tooth disease type 4D and 4G (OMIM 601455 and 605285), Congenital cataract facial dysmorphism neuropathy (OMIM 604166), Gitelman syndrome (OMIM 263800), and Galactokinase deficiency (OMIM 230200) [41,42]. In light of the genetic specificity found in the Roma, we investigated the frequency of two identified *ABCG2* mutants (c.393G>T and c.706C>T) in our control cohort of 60 subjects of Roma origin (see Section 2.2 in Results). MAFs of c.393G>T in the Roma cohort and European population were 0.833% (1 allele/120 subjects) and 0.002% (6 alleles/64,397 subjects), respectively. Although the sample size of our control cohort was very modest, it could be large enough to imply that the origin of c.393G>T might be the Roma population. On the other hand, the prevalence of c.706C>T was higher in Europeans than in the Roma, suggesting that these two *ABCG2* mutations could have different genetic origins.

For clinical practice, our findings suggest a need for further discussion about the potential benefits of urate-lowering therapy after a diagnosis of hyperuricemia in pediatriconset patients with ABCG2 dysfunction. Interestingly, harboring p.Q141K is reportedly associated with inadequate response to allopurinol (characterized by a smaller reduction in serum urate concentrations compared with WT) [29,43–45]; allopurinol, which inhibits uric acid production, is a well-known and widely used drug for lowering SUA. Although the mechanisms of action for the inadequate response are still unclear, other SUA-lowering drugs may be somewhat better in terms of efficacy for patients with a dysfunctional *ABCG2* allele, which puts them at higher risk of developing hyperuricemia/gout. On the other hand, among the clinically-used inhibitors for the production of uric acid (i.e., allopurinol, febuxostat, and topiroxostat), only febuxostat, to the best of our knowledge, strongly and clinically inhibits ABCG2 function as a urate transporter [35]. This is also supported by a recent clinical study that showed orally-administered febuxostat inhibits intestinal ABCG2 in humans [46]. In this context, the efficacy of febuxostat as an SUA-lowering drug can be partially blocked by ABCG2 inhibition, except in cases of completely null ABCG2 function. Considering this complexity, as well as risks of adverse effects [47], the best SUA-lowering drugs, including uricosuric agents and uric acid production inhibitors, should be carefully determined for each patient.

Additionally, we can emphasize the clinical importance of the documentation regarding the family we addressed in this study, given the infrequency of detailed studies on SUA levels in children. Indeed, pediatric-onset of hyperuricemia is relatively rare in clinical practice; it is often associated with rare conditions (e.g., purine metabolic disorders; kidney disorders including uromodulin-associated disorders; metabolic genetic disorders including glycogen storage disease, hereditary fructose intolerance, and mitochondrial disorders). As we showed previously [48], the levels of SUA and FE-UA are quite dynamic in the first year of life. In brief, the SUA levels are low in infancy (131–149 µmol/L; 2.2–2.5 mg/dL at 2–3 months of age) due to the high FE-UA levels (>10%); the FE-UA levels decrease to approximately 8% at age 1 and then stay through childhood, which is associated with mean SUA levels (208–268 µmol/L; 3.5–4.5 mg/dL) of children. At adolescence (after age 12), the FE-UA levels significantly decrease in boys but not in girls, resulting in a further significant increase in SUA levels in young men but not in young women. These pieces of information support the rarity of our case of which proband is a 12-year-old girl

with chronic asymptomatic hyperuricemia characterized by elevated serum urate (397–405 µmol/L; 6.67–6.81 mg/dL) and the decreased FE-UA (2.2–3.5%).

Some limitations warrant mention. It is unclear whether there could have been other genetic factors also affecting the early-onset phenotypes in the family we studied, although harboring dysfunctional *ABCG2* mutations is the most plausible explanation, as the study showed. A previous study showed that in the context of extra-renal underexcretion hyperuricemia, a genetic dysfunction of ABCG2 increased SUA levels and apparent urinary urate excretion, which was coupled with decreased intestinal urate excretion. Thus, we can assume the presence of latent mechanisms causing the decreased (<5%) FE-UA levels observed in our patients. Although there is little information available regarding this, a possible factor could be increased renal urate reabsorption, which can be mediated by up-regulation of renal urate re-absorbers, including *URAT1/SLC22A12, GLUT9*/*SLC2A9*, and *organic anion transporter 10* (known as *SLC22A13*) [49]. In this context, genetic variations affecting the expression of such genes will be the targets of future studies.

In conclusion, we found a representative case of pediatric hyperuricemia with familial gout that harbored two dysfunctional *ABCG2* mutations. Genetic variations in *ABCG2* should be kept in mind during diagnostic procedures for pediatric-onset hyperuricemia. Considering *ABCG2* genotypes will be beneficial for patients with early-onset and/or familial hyperuricemia and gout. This type of genetic information will also allow a personalized approach regarding the best urate-lowering treatment (i.e., uric acid production inhibitors or uricosuric agents) for patients with dysfunctional ABCG2 variants, as well as the best time to initiate pharmacotherapy for hyperuricemia.
