*2.1. Subjects*

The clinical and biochemical data from this study are summarized in Table 1. Repeated biochemical analysis of the proband (a 12-year-old girl with chronic asymptomatic hyperuricemia) showed elevated serum urate (397–405 µmol/L; 6.67–6.81 mg/dL) with a decreased fractional excretion of uric acid (FE-UA) (2.2–3.5%). No clinical or laboratory symptoms of renal disease were present in the patient. The girl's mother (38 years-old) had previously presented with asymptomatic hyperuricemia (420–439 µmol/L; 7.06–7.38 mg/dL of serum urate); both the maternal grandfather (66-years-old) and the maternal uncle (40-years-old) had presented with hyperuricemia (500–537 µmol/L; 8.41– 9.03 mg/dL) and early-onset gout, with the first gout attack occurring in the uncle when he was 35-years-old. The girl's father (38-years-old) presented with asymptomatic hyperuricemia (487 µmol/L; 8.19 mg/dL of serum urate), which seemed to be associated with metabolic syndrome (i.e., hypertriglyceridemia, hypertension, and central obesity). Metabolic investigation for purine metabolism (i.e., hypoxanthine and xanthine levels in the urine) found normal urinary excretion, suggesting that hyperuricemia in our patients and family relatives was not caused by an excess production of uric acid. Thus, we focused on the physiologically important urate transporters, especially ABCG2, which regulate urate handling in the body as described below.


**Table 1.** Clinical and biochemical data of a family with early-onset hyperuricemia and gout.

HA, hyperuricemia; SUA, serum uric acid; UUA, urine uric acid; FE-UA, fractional excretion of uric acid; T2D, type 2 diabetes mellitus; n.d., not determined. Reference ranges are as follows. SUA: 120–360 µmol/L (2.02–6.05 mg/dL) (< 15 years and female), 120–420 µmol/L (2.02–7.06 mg/dL) (male); UUA: 0.1–1.0 mmol/mol creatinine (< 15 years), 0.1–0.8 mmol/mol creatinine (≥15 years); FE-UA: 5–20% (<13 years), 5–12% (male), 5–15% (female).

### *2.2. Genetic Analysis 2.2. Genetic Analysis*

**SUA during treatment [µmol/L (mg/dL)]** 

Targeted exon sequencing analyses of *ABCG2* revealed that the proband had two rare heterozygous non-synonymous mutations, i.e., (1) c.393G>T (p.M131I, rs759726272) and (2) c.706C>T (p.R236X, rs140207606). No other exonic mutations were found, including previously characterized common genetic risk factors for hyperuricemia/gout, such as *ABCG2* c.376C>T (p.Q126X) and c.421C>A (p.Q141K). Moreover, a segregation analysis of the family confirmed the two mutations as probably being disease-associated mutations. Heterozygous c. 393G>T and c.706C>T were identified through the maternal line in the mother, uncle, and grandfather of the proband. The pedigree is shown in Figure 1 along with representative electropherograms of partial *ABCG2* sequences showing a heterozygous missense mutation (c.393G>T) in exon 5 and a nonsense mutation (c.706C>T) in exon 7. Moreover, sequencing analyses, including analyses of previously known SUAassociating loci, found no disease-associated non-synonymous mutations in *GLUT9* and *URAT1*. Although two mutations, c.73G>A (p.G25R, rs2276961) and c.844G>A (p.V282I, rs16890979), were found in *GLUT9*; however, previous studies have shown that they would not cause hyperuricemia/gout [30,31]. Targeted exon sequencing analyses of *ABCG2* revealed that the proband had two rare heterozygous non-synonymous mutations, i.e., (1) c.393G>T (p.M131I, rs759726272) and (2) c.706C>T (p.R236X, rs140207606). No other exonic mutations were found, including previously characterized common genetic risk factors for hyperuricemia/gout, such as *ABCG2* c.376C>T (p.Q126X) and c.421C>A (p.Q141K). Moreover, a segregation analysis of the family confirmed the two mutations as probably being disease-associated mutations. Heterozygous c. 393G>T and c.706C>T were identified through the maternal line in the mother, uncle, and grandfather of the proband. The pedigree is shown in Figure 1 along with representative electropherograms of partial *ABCG2* sequences showing a heterozygous missense mutation (c.393G>T) in exon 5 and a nonsense mutation (c.706C>T) in exon 7. Moreover, sequencing analyses, including analyses of previously known SUA-associating loci, found no disease-associated non-synonymous mutations in *GLUT9* and *URAT1*. Although two mutations, c.73G>A (p.G25R, rs2276961) and c.844G>A (p.V282I, rs16890979), were found in *GLUT9*; however, previous studies have shown that they would not cause hyperuricemia/gout [30,31].

*Int. J. Mol. Sci.* **2021**, *22*, x FOR PEER REVIEW 4 of 15

n.d. n.d.

years); FE-UA: 5–20% (< 13 years), 5–12% (male), 5–15% (female).

**Therapy** No No

**FE-UA [%]** 2.2 4.6 2.9 n.d. 3.9

HA, hyperuricemia; SUA, serum uric acid; UUA, urine uric acid; FE-UA, fractional excretion of uric acid; T2D, type 2 diabetes mellitus; n.d., not determined. Reference ranges are as follows. SUA: 120–360 μmol/L (2.02–6.05 mg/dL) (< 15 years and female), 120–420 μmol/L (2.02–7.06 mg/dL) (male); UUA: 0.1–1.0 mmol/mol creatinine (< 15 years), 0.1–0.8 mmol/mol creatinine (≥15

Allopurinol (100 mg per

> 446 (7.50) (noncompliance)

Colchicine; Allopurinol (300 mg per day)

422 (7.09) (noncompliance) No

n.d.

day)

**Figure 1.** Identification of novel disease-associated mutations c.393G>T (p.M131I) and c.706C>T (p.R236X) in *ABCG2*. Left, the pedigree of a Czech family with early-onset hyperuricemia and gout; Right, representative electropherograms of partial sequences of *ABCG2* showing the heterozygous point mutations (red) discovered in our present study. I–III, generation of the family; P, proband; y, years old. **Figure 1.** Identification of novel disease-associated mutations c.393G>T (p.M131I) and c.706C>T (p.R236X) in *ABCG2*. Left, the pedigree of a Czech family with early-onset hyperuricemia and gout; Right, representative electropherograms of partial sequences of *ABCG2* showing the heterozygous point mutations (red) discovered in our present study. I–III, generation of the family; P, proband; y, years old.

Further sequencing analyses revealed that neither of these *ABCG2* mutations was detected in a previously enrolled cohort of 360 patients with hyperuricemia/gout and a co-Further sequencing analyses revealed that neither of these *ABCG2* mutations was detected in a previously enrolled cohort of 360 patients with hyperuricemia/gout and a cohort of 132 normouricemia control subjects of European descendent [32]. However, in another control group of 60 subjects of Roma ethnicity [33], although serum urate and FE-UA values were not available, we found one copy of c.393G>T (p.M131I), but without c.706C>T (p.R236X), in a male hemodialysis patient with severe chronic kidney disease. This finding suggested that c.393G>T (p.M131I) and c.706C>T (p.R236X) can occur independently, which led us to examine the effect of each non-synonymous mutation on the ABCG2 protein.

Additionally, the independent genetic origin of these two *ABCG2* mutations was also strongly supported by supplementary analyses of MAF data that is openly available from the Exome Aggregation Consortium (http://exac.broadinstitute.org/ (accessed on 30 November 2020)). With regard to c.393G>T (p.M131I), only six heterozygotes of the minor allele (393T) were found in a cohort of 64,397 European subjects (MAF, 0.0047%); with even fewer carriers in other populations (total worldwide MAF frequency = 0.0021%). With regard to c.706C>T (p.R236X), 48 heterozygotes of the minor allele (706T) were found in a cohort of 645,050 European subjects (MAF = 0.0372%, which is almost 8 times greater

than the MAF of c.393G>T); in other populations, frequencies ranging from 0.0000% in European Finnish to 0.1254% (the highest) in Ashkenazi Jewish were found.
