*4.2. Clinical Investigations and Sequence Analyses*

Urate and creatinine levels were measured as described previously [51] using a specific enzymatic method and the Jaffé reaction, which was adapted for an auto-analyzer (Hitachi Automatic Analyzer 902; Roche, Basel, Switzerland). Metabolic investigations of purine metabolism (hypoxanthine and xanthine levels in urine) were also conducted using a method established in a previous study [51]. The proband was screened for metabolic (e.g., glycogen storage disease, hereditary fructose intolerance, and mitochondrial disorders) and kidney disease associated with hyperuricemia (e.g., uromodulin-associated disorders), using our previously published diagnostic algorithm [29].

All coding regions and exon-intron boundaries in *ABCG2* and *GLUT9/SLC2A9,* and exons 7 and 9 in *URAT1/SLC22A12* were analyzed from genomic DNA, as described previously [29,33,52]. The reference sequence for *ABCG2* was defined as version ENST00000237612.7 (location: Chromosome 4: 88,090,269–88,158,912 reverse strand) (www.ensembl.org (accessed on 30 November 2020)). For *GLUT9/SLC2A9* (NM\_020041.2; NP\_064425.2; SNP source dbSNP 132) and *URAT1/SLC22A12* (NM\_144585.3), the reference genomic sequence was defined as version NC\_000004.12 (Chromosome 4: 9,771,153–10,054,936) and NC\_000011.8 (Chromosome 11: 64,114,688–64,126,396), respectively.

It is worth special mention that the world's highest frequency of the main dysfunctional variants of URAT1, p.T467M (MAF, 5.6%) and p.L415\_G417del (MAF, 1.9%), was recently identified in a Roma population (1,016 individuals) from specific regions of the Czech Republic, Slovakia, and Spain [33,53]. According to MAF data from the Exome Aggregation Consortium, p.T467M (rs200104135) showed only one heterozygous allele in a cohort of 15,296 in a South Asian population (MAF, 0.003%) and no occurrence in other ethnic populations; no occurrence of the p.L415\_G417del allele was seen in the whole population, which supports the Roma-specific prevalence of these two URAT1 variants. For this reason, we looked for both URAT1 variants and confirmed that neither variant was present in our studied family.
