## 4 Discussion and Conclusions
Variant p.R325W was localized within the putative extracellular loop. This variant has not yet been identified in the patients of those with renal hypouricemia, but the nature of this mutation strongly suggests that it is pathogenic; PolyPhen software ([[http://genetics.bwh.harvard.edu/pph/](http://genetics.bwh.harvard.edu/pph/)]([http://genetics.bwh.harvard.edu/pph/](http://genetics.bwh.harvard.edu/pph/))) suggested that the variant is possibly damaging (score 0.72). Moreover, another predictive software, SIFT ([[http://sift.jcvi.org/](http://sift.jcvi.org/)]([http://sift.jcvi.org/](http://sift.jcvi.org/))), suggests that this variant is deleterious (score 0). On the other hand, CADD ([[https://cadd.gs.washington.edu/](https://cadd.gs.washington.edu/)]([https://cadd.gs.washington.edu/](https://cadd.gs.washington.edu/))), REVEL, and MetaLR predictive software indicate that the impact of the variant is likely to be tolerated or benign. In the middle stands Mutation Assessor ([[http://mutationassessor.org/r3/](http://mutationassessor.org/r3/)]([http://mutationassessor.org/r3/](http://mutationassessor.org/r3/))) which predicts a moderate functional impact. Evolutionary analysis of URAT1 paralogs, including six mammalian species, revealed conservation of p.R325W only between human and chimpanzee (Figure 2). Human and Simian monkeys possess high affinity and low capacity URAT1 transporter which diverged from the original low affinity, high capacity paralog (mouse, rat, horse and dog) 43 MYA [20]. Authors described four key amino acid substitutions in human URAT1 positions 25, 27, 365 and 414 as a crucial for the high to low affinity and low to high capacity shift. Similarly, as a variant p.R325W, all four amino acid residues are conserved between human and baboon, but not among other mammalian species. The functional characterization of p.R325W showed significantly decreased urate uptake and a weak, discontinuous URAT1 signal on the plasma membrane and reduced intracytoplasmic staining. The results suggested that p.R325W variant may not affect URAT1 function qualitatively (via alteration of its intrinsic transporter activity), but rather do so quantitatively (via decreasing its cellular protein level). Taken together, the data confirm the causality of the p.R325W variant relative to renal hypouricemia 1. Detailed investigations of serum uric acid concentrations and excretion fractions of uric acid in patients with unexplained hypouricemia are needed. Many patients with RHUC may be asymptomatic; however, pediatric nephrologists know that RHUC can cause acute renal failure, especially after strenuous physical activity. Another risk of RHUC is the development of nephrolithiasis. Although renal hypouricemia is a rare hereditary disorder, the frequency of novel URAT1 associated variants shows that this condition is underdiagnosed. RHUC should be considered not only in patients from Japan or Asia. The phenotypic severity of RHUC1 is not correlated with results from functional characterizations of URAT1 variants. Functional studies regarding the impact of novel associated variants are necessary to determine their correlation with scores from prediction algorithms and to confirm causality. Conceptualization, B.S. ; validation, B.S. ; [PERSON] conducted sequencing analyses; [PERSON], [PERSON], [PERSON] and [PERSON] worked on experiments using _Xenopus_ oocytes, and analyzed the data; [PERSON] was responsible for clinical observations; data curation, B.S. ; writing, B.S. ; project administration, B.S. ; funding acquisition, B.S. Supported by the Ministry of Health of the Czech Republic: AZV 15-26693A, the project for conceptual development of research organization 00023728 (Institute of Rheumatology) and RVO VFN64165. The authors declare no conflict of interest. Figure 2: Alignment of the p.R325W URAT1 amino acids in the studied allelic variants with chimpanzee, horse, dog, rat and mouse paralogs.