Idiopathic infantile hypercalcemia (IIH) is characterized by severe hypercalcemia with failure to thrive, vomiting, dehydration, and nephrocalcinosis. Recently, mutations in the vitamin D catabolizing enzyme 25-hydroxyvitamin D 3 -24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH) 2 D 3 . In a subgroup of patients who presented in early infancy with renal phosphate wasting and symptomatic hypercalcemia, mutations in CYP24A1 were excluded. Four patients from families with parental consanguinity were subjected to homozygosity mapping that identified a second IIH gene locus on chromosome 5q35 with a maximum logarithm of odds (LOD) score of 6.79. The sequence analysis of the most promising candidate gene, SLC34A1 encoding renal sodium-phosphate cotransporter 2A (NaPi-IIa), revealed autosomal-recessive mutations in the four index cases and in 12 patients with sporadic IIH. Functional studies of mutant NaPi-IIa in Xenopus oocytes and opossum kidney (OK) cells demonstrated disturbed trafficking to the plasma membrane and loss of phosphate transport activity. Analysis of calcium and phosphate metabolism in Slc34a1-knockout mice highlighted the effect of phosphate depletion and fibroblast growth factor-23 suppression on the development of the IIH phenotype. The human and mice data together demonstrate that primary renal phosphate wasting caused by defective NaPi-IIa function induces inappropriate production of 1,25-(OH) 2 D 3 with subsequent symptomatic hypercalcemia. Clinical and laboratory findings persist despite cessation of vitamin D prophylaxis but rapidly respond to phosphate supplementation. Therefore, early differentiation between SLC34A1 (NaPi-IIa) and CYP24A1 (24-hydroxylase) defects appears critical for targeted therapy in patients with IIH.
BACKGROUND Inherited metabolic disorders associated with nephrocalcinosis are rare conditions. The aim of this study was to identify the genetic cause of an Israeli-Arab boy from a consanguineous family with severe nephrocalcinosis and kidney insufficiency. METHODS Clinical and biochemical data of the proband and family members were obtained from both previous and recent medical charts. Genomic DNA was isolated from peripheral blood cells. The coding sequence and splice sites of candidate genes (CYP24A1, CYP27B1, FGF23, KLOTHO, SLC34A3 and SLC34A1) were sequenced directly. Functional studies were performed in Xenopus laevis oocytes and in transfected opossum kidney (OK) cells. RE-SULTS Our patient was identified as having nephrocalcinosis in utero, and at the age of 16.5 years, he had kidney insufficiency but no bone disease. Genetic analysis revealed a novel homozygous missense mutation, Arg215Gln, in SLC34A1, which encodes the renal sodium phosphate cotransporter NaPiIIa. Functional studies of the Arg215Gln mutant revealed reduced transport activity in Xenopus laevis oocytes and increased intracellular cytoplasmic accumulation in OK cells. CONCLUSIONS Our findings show that dysfunction of the human NaPiIIa causes severe renal calcification that may eventually lead to reduced kidney function, rather than complications of phosphate loss.
Homeostasis of inorganic phosphate (Pi) is essential for various physiologic functions. The sodium phosphate cotransporter NaPi‐IIa is located in the brush border membrane (BBM) of the renal proximal tubule and represents the major renal Pi transporter. The plasma concentration of Pi is determined by factors regulating NaPi‐IIa abundance at the BBM. We are developing a novel mouse model expressing NaPi‐IIa tagged with red fluorescent protein (RFP) to analyze the regulation of the cotransporter at the BBM in real time and space and at the molecular level. We have generated RFP‐NaPi‐IIa constructs containing the RFP at three different locations of the cotransporter, the very N‐terminus, within the N‐terminal tail and within the big extracellular loop. Control experiments in opossum kidney cells and in Xenopus laevis oocytes to analyze the cotransporter polarization, PTH sensitivity, Na+‐dependent cotransport activity and kinetic characterization revealed that the RFP within the N‐terminal tail did not impair any of the analyzed parameters. Upon identification of the proper insertion site, we generated three pairs of transcription activator‐like effector nucleases (TALENs) predicted to cut the Slc34a1 (NaPi‐IIa) gene within the desired sequence. The pair with the highest cutting efficiency was chosen for the generation of RFP‐NaPi‐IIa mice. Consequently, we produced RFP donor DNA containing 5' and 3' homologous arms, as well as the RFP coding sequence. TALENs and the RFP donor were injected into one‐cell stage embryos to generate transgenic mice.This work was supported by NCCR Kidney.CH
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