*2.3. Functional Analysis*

To investigate the effect of the two identified non-synonymous mutations (p.M131I and p.R236X) on the intracellular processing and function of the ABCG2 protein, we conducted several biochemical analyses using transiently ABCG2-expressing mammalian cells (Figure 2). First, immunoblotting for *N*-glycosidase (PNGase F)-treated whole cell lysates (Figure 2A) demonstrated that p.M131I had a minimal effect on protein levels and *N*-linked glycosylation status, while the p.R236X variant was detected as truncated forms of the protein with weaker band intensities compared to ABCG2 wild-type (WT), as would be expected based on its amino acid sequence. Confocal microscopy (Figure 2B) showed that, like ABCG2 WT, the p.M131I variant was mainly located on the plasma membrane, while the p.R236X variant exhibited little plasma membrane localization. Expression of the p.M131I variant as a glycoprotein on plasma membrane-derived vesicles was confirmed using immunoblotting (Figure 2C). Next, using a vesicle transport assay [34] with an optimized experimental procedure for determining the initial rate of urate transport by ABCG2 based on our previous studies [12,35], ABCG2 function was evaluated as having ATP-dependent urate transport activity into plasma membrane vesicles (Figure 2D). The functional assay revealed that contrary to the WT, p.M131 had limited ATP-dependent urate transport activity, with the ABCG2-mediated urate transport activity of this variant calculated to be 14 ± 2% of the WT controls. The p.R236X variant was functionally null (Figure 2D), which was consistent with the results of our biochemical analyses (Figure 2A–C). *Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 6 of 15

**Figure 2.** Functional characterization of disease-associated ABCG2 mutations. (**A**) Immunoblot of whole cell lysate samples. α-Tubulin used as the loading control. WT, wild-type. (**B**) Confocal microscopy of intracellular localization. Nuclei were stained with TO-PRO-3 iodide (gray). Bars, 10 μm. (**C**) Immunoblot of plasma membrane vesicles. Na<sup>+</sup> /K<sup>+</sup> ATPase as the loading control. (**D**) Urate transport activity. The incubation condition was 37 °C for 10 min in the presence of 20 μM radiolabeled urate. Data are expressed as the mean ± SD. *n* = 3–4. ††, *P* < 0.01; NS, not significantly different between groups (two-sided *t*-test). **Figure 2.** Functional characterization of disease-associated ABCG2 mutations. (**A**) Immunoblot of whole cell lysate samples. α-Tubulin used as the loading control. WT, wild-type. (**B**) Confocal microscopy of intracellular localization. Nuclei were stained with TO-PRO-3 iodide (gray). Bars, 10 µm. (**C**) Immunoblot of plasma membrane vesicles. Na+/K<sup>+</sup> ATPase as the loading control. (**D**) Urate transport activity. The incubation condition was 37 ◦C for 10 min in the presence of 20 µM radiolabeled urate. Data are expressed as the mean ± SD. *n* = 3–4. ††, *P* < 0.01; NS, not significantly different between groups (two-sided *t*-test).

tion between hyperuricemia/gout and the *ABCG2* gene.

ruricemia 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 associa-

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

**3. Discussion** 
