Figure 1.
Identification of mutations within SLC22A12 encoding URAT1.
A. Chromatograms showing sequence data and translated amino acids. These demonstrate heterozygous missense variants (above, arrowed) and normal controls (below). B. SLC22A12 encodes URAT1, a 553 amino acid protein with a predicted 12 Transmembrane domains (TMPred). It has an intracellular N- and C- terminus. Amino acid residues implicated in hypouricaemia and modelled in the present study are highlighted in red and include Isoleucine, I at position 75, arginine, R at position 347, Valine V at position 388 and Arginine, R at position 434.
Table 1.
Demographic, clinical, biochemical and molecular genetic data on 6 patients with renal hypouricaemia.
Table 2.
URAT1 variants and their in silico analysis for conservation and pathogenicity.
Figure 2.
Functional analysis of changes in URAT1 expressed in HEK293 cells.
A. HEK293 cells were transiently transfected with Flag-tagged URAT1 cDNA constructs (wild-type and variants I75T, R347S, V388M, R434C and R434H). Plasma membrane expression was detected using an anti-FLAG monoclonal antibody, secondarily detected using an Alexa Fluor® 488 (green) antibody. Nuclei are counter stained using DAPI (blue). B. Uric acid uptake by HEK293 cells transiently transfected with wild-type URAT1 or its mutants was measured using [14C]Urate at 2 min, at 37°C and pH7.4. Significant reductions in urate transport activity was seen in some of the disease-associated variants. Data are mean ± S.E.M with n = 4. ***, P<0.001 when compared with wild type.