Autosomal-Recessive Mutations in SLC34A1 Encoding Sodium-Phosphate Cotransporter 2A Cause Idiopathic Infantile Hypercalcemia

Abstract

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 D3-24-hydroxylase (CYP24A1) were described that lead to increased sensitivity to vitamin D due to accumulation of the active metabolite 1,25-(OH)2D3. 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)2D3 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.

Keywords: activated Vitamin D; genetic renal disease; hypercalciuria; mineral metabolism; molecular genetics.

Figure 1.

Integrated scheme of calcium and phosphate metabolism. The activation of vitamn D to its biologically active form 1,25-(OH)₂D₃ by 1α-hydroxylase (CYP27B1) as well as its degradation by 24-hydroxylase (CYP24A1) are tightly controlled by 1,25-(OH)₂D₃ itself, serum calcium, and PTH (lower part). In addition, vitamin D metabolism is critically influenced by phosphate homeostasis via the action of the primary phosphaturic hormone FGF23 that limits the action of active 1,25-(OH)₂D₃ by inhibiting 1α-hydroxylase (CYP27B1) and activating 24-hydroxylase (CYP24A1) (upper part).

Figure 2.

Genetic and functional analyses of SLC34A1/NaPi-IIa. (A) Identified mutations in the SLC34A1 gene. In total, 16 different mutations were identified including six missense mutations, two frame-shift mutations, one in-frame deletion, two stop mutations, and five splice-site mutations. (B) Secondary topology of the human NaPi-IIa protein (adapted from Fenollar-Ferrer et al.³⁶) with mutations indicated. (C) Phosphate transport activity of wild-type and mutant hNaPi-IIa. Uptakes were performed in Xenopus oocytes 3 days after injection of cRNA encoding hNaPi-IIa. n=2, each 8–10 oocytes; NI, non-injected; wt, wild-type. (D) Expression of human NaPi-IIa cotransporters in OK cells. Cells were transfected with pEGFP plasmids containing either wild-type or mutant hNaPi-IIa, as well as with the empty pEGFP plasmid. Confluent cultures were processed for confocal microscopy. (a) Focal planes of lateral projections. (b) Focal planes of apical projections. (c) Cross-sections. NaPi-IIa signal is shown in green and the actin staining in red.

Figure 3.

Clinical course of patient F9.1 during acute disease manifestation. Whereas rehydration and omitting of vitamin D prophylaxis did not lead to correction of hypercalcemia and clinical improvement, phosphate supplementation implemented after genetic diagnosis of SLC34A1 mutations resulted in normophosphatemia, a normalization of calcium metabolism, a reduction in calcium excretion, as well as a rapid clinical recovery and weight gain. The inset shows severe medullary nephrocalcinosis on renal ultrasonography in this infant.

Figure 4.

Biochemical data of Slc34a1 knockout mice in comparison to wild-type animals. Both mice were fed diets with low or high phosphate content (lowP/highP) and vitamin D (lowD/highD), respectively. HighD diets resulted in a vitamin D overload in both knockout and wild-type mice. Wild-type mice adequately limited vitamin D activation by downregulating Cyp27b1 and activating Cyp24a1 expression. In contrast, phosphate-depleted Slc34a1 knockout mice exhibited low FGF23 levels, provoking a reverse regulation of Cyp27b1 (1α-hydroxylase) and Cyp24a1 (24-hydroxylase) expression. Consequently, these mice were not able to limit vitamin D activation, leading to an aggravation of hypercalcemia. Dysregulated calcium homeostasis in these knockout mice was only slightly improved by limiting vitamin D supply with persistence of hypercalcemia and hypercalciuria. In contrast, high phosphate supplementation restored serum levels of phosphate and FGF23 enabling a normalization of 1,25-(OH)₂D₃ and serum calcium levels. Significant differences between knockout and wild-type mice under lowP/highD diet are indicated by bold letters, significance levels are: *P<0.05; **P<0.01; ***P<0.001; # = normalized Cyp27b1 and Cyp24a1 expression levels (for details see Supplemental Material).

Figure 5.

Pathophysiology of disturbed NaPi-IIa function in the proximal renal tubule. (A) Under physiologic conditions, proximal tubular phosphate reabsorption via NaPi-IIa (and NaPi-IIc, not shown) ensures the maintenance of phosphate homeostasis. Phosphate reabsorption via NaPi-IIa is limited by the concerted action of PTH (not shown) and FGF23. Besides its phosphaturic effect, FGF23 negatively regulates the action of 1,25-(OH)₂D₃ by inhibiting the expression of 1α-hydroxylase (CYP27B1) and activating 24-hydroxylase (CYP24A1). (B) As a consequence of a NaPi-IIa defect, phosphate depletion leads to a decrease of FGF23 levels. In turn, an unrestricted activation of 1,25-(OH)₂D₃ results in hypercalcemia, hypercalciuria, and nephrocalcinosis.