Novel allelic variants and evidence for a prevalent mutation in URAT1 causing renal hypouricemia: biochemical, genetics and functional analysis

Abstract

Renal hypouricemia (RHUC) is a heterogeneous inherited disorder characterized by impaired tubular uric acid (UA) transport with severe complications, such as acute kidney injury (AKI). Type 1 is caused by a loss-of-function mutation in the SLC22A12 gene (URAT1), type 2 in the SLC2A9 gene (GLUT9). This article describes three Czech families with RHUC type 1. The serum UA in the probands was 0.9, 1.1 and 0.5 mg/dl and expressed as an increase in the fractional excretion of UA (48, 43 and 39%). The sequencing analysis of SLC22A12 revealed three novel variants: p.G366R, p.T467M and a deletion p.L415_G417del. A detailed metabolic investigation in proband C for progressive visual failure supported suspicion of neuronal ceroid lipofuscinosis type 7 conditioned by the mutation in the MFSD8 gene. Functional studies showed significantly decreased urate uptake and a mis-localized URAT1 signal in p.G366R, p.L415_G417del and p.T467M. Furthermore, colocalization studies showed accumulation of URAT1 protein in the endoplasmic reticulum. The findings suggest that loss-of-function mutations cause RHUC via loss of UA absorption partly by protein misfolding. However, they do not necessarily lead to AKI and a possible genotype-phenotype correlation was not proposed. Furthermore, results confirm an uneven geographical and ethnic distribution of SLC22A12 variants; the p.L415_G417del mutation predominates in the Roma ethnic group in the Czech Republic.

Figure 1

Urate transport ability of the URAT1 mutants found in the patients. Urate uptakes by the oocyte-expressed URAT1 mutants except p.R477H were significantly decreased compared with oocytes injected with wild-type (wt) URAT1 cRNA (*P<0.05, **P<0.01, ***P<0.001). p.R477H mutant showed the same urate-uptake ability as wt URAT1. No injection represents non-cRNA-injected oocytes. n=6 (no injection, wt) and 3 (p.G366R, p.R477H, p.del415-417, p.T467M).

Figure 2

Immunocytochemical analysis of oocytes injected with 50 ng of cRNA encoding the wild-type (wt) or mutant URAT1 performed with anti-URAT1 polyclonal antibody. The signal of URAT1 is green, autofluorescent granules in cytoplasm of oocytes give a blue signal. (a) Water-injected oocyte without any detectable URAT1 signal. (b, c) Oocytes injected with the wt cRNA (b) or with cRNA of p.R477H (c) exhibited a strong linear signal on the plasma membrane and a finely granular intracytoplasmic signal. (d, e) The mutant p.G366R (d) and p.L415_G417del (e) were characterized by the lack of plasma membrane expression of URAT1. Cytoplasmic staining intensity was lower than in the wt or p.R477H. Note the tendency of intracytoplasmic signal accumulation in p.G366R (d). Staining of mutant p.T467M was faint and irregular on the plasma membrane and reduced in cytoplasm (f). Plasma membrane of oocytes is marked by arrows.

Figure 3

Colocalization of URAT1 mutant variants with endoplasmatic reticulum (ER). Strong colocalization with ER is observed in p.G366R. URAT1 localization in wild-type (wt) and p.R477H is predominantly in cytoplasmic membrane. Dispersed signal is also seen in cytoplasm. Partial colocalization with ER is visible in p.R477H mutant. In wt the URAT1 and ER signals are rather separated. Scale bar represents 50 μm.

Figure 4

URAT1 allelic variants. Alignment of the URAT1 amino acids in the studied allelic variants with chimpanzee, horse, dog, mouse, rat and Xenopus paralogs.