SLC34A3 mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria predict a key role for the sodium-phosphate cotransporter NaPi-IIc in maintaining phosphate homeostasis

Abstract

Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare disorder of autosomal recessive inheritance that was first described in a large consanguineous Bedouin kindred. HHRH is characterized by the presence of hypophosphatemia secondary to renal phosphate wasting, radiographic and/or histological evidence of rickets, limb deformities, muscle weakness, and bone pain. HHRH is distinct from other forms of hypophosphatemic rickets in that affected individuals present with hypercalciuria due to increased serum 1,25-dihydroxyvitamin D levels and increased intestinal calcium absorption. We performed a genomewide linkage scan combined with homozygosity mapping, using genomic DNA from a large consanguineous Bedouin kindred that included 10 patients who received the diagnosis of HHRH. The disease mapped to a 1.6-Mbp region on chromosome 9q34, which contains SLC34A3, the gene encoding the renal sodium-phosphate cotransporter NaP(i)-IIc. Nucleotide sequence analysis revealed a homozygous single-nucleotide deletion (c.228delC) in this candidate gene in all individuals affected by HHRH. This mutation is predicted to truncate the NaP(i)-IIc protein in the first membrane-spanning domain and thus likely results in a complete loss of function of this protein in individuals homozygous for c.228delC. In addition, compound heterozygous missense and deletion mutations were found in three additional unrelated HHRH kindreds, which supports the conclusion that this disease is caused by SLC34A3 mutations affecting both alleles. Individuals of the investigated kindreds who were heterozygous for a SLC34A3 mutation frequently showed hypercalciuria, often in association with mild hypophosphatemia and/or elevations in 1,25-dihydroxyvitamin D levels. We conclude that NaP(i)-IIc has a key role in the regulation of phosphate homeostasis.

Figure 1

Genealogical, clinical, and genetic findings in a previously published Bedouin kindred with HHRH. A, Partial pedigree of the Bedouin kindred. Blackened circles or squares indicate individuals who developed rickets during childhood, along with renal phosphate wasting, hypophosphatemia, and hypercalciuria. Unblackened symbols without a question mark (?) indicate individuals who were healthy; the question mark indicates unknown phenotype. A symbol with only the right upper quadrant blackened indicates an individual affected with only hypercalciuria. Samples for individuals with symbols with dashed outlines on the gray background were unavailable for genotyping. B, Haplotypes for chromosome region 9q34 between markers D9S1826 and D9S1838. Alleles for microsatellite markers are designated in bp or are coded; for the c.228delC mutation, “+” and “−” indicate the presence and absence of the deletion, respectively. The haplotype associated with HHRH is depicted by numbers on a gray background; regions that were excluded because of inferred ancestral recombination events are shown by numbers on a white background that are delineated with a horizontal black line. C, Endonuclease digestion to identify the c.228delC mutation. The deletion abolishes an StuI site. Therefore, PCR-amplified DNA from the mutant allele (451 bp) could not be digested with this endonuclease. In contrast, DNA amplified from the wild-type allele yielded 359- and 92-bp fragments (see also table 2).

Figure 2

Genetic findings in three previously published kindreds with HHRH (A, C, and D). In the pedigrees, the use of symbols and shading is the same as in figure 1 unless otherwise indicated. Haplotypes associated with HHRH are depicted by numbers on a dark-gray or light-gray background. Mutations were identified by nucleotide sequence analysis and were confirmed by restriction endonuclease digestion of the appropriate PCR products when possible (see table 2). Note that, in kindred D, c.1579_1581del cannot be unambiguously phased.

Figure 3

Location and type of SLC34A3 mutations identified in four kindreds with HHRH. Upper panel, Predicted NaP_i-IIc, with eight membrane-spanning domains. The approximate locations of point mutations are shown in black letters on a white background if they affect identical amino acid residues in the NaP_i-IIc molecules of human, mouse, rat, and dog; they are depicted in white letters on a black background if they affect amino acid residues that are identical in all type II sodium-phosphate cotransporters. The c.228delC deletion (black arrow) is predicted to lead to truncation of NaP_i-IIc within the first membrane-spanning domain. Lower panel, Partial sequence of human NaP_i types IIa, IIb, and IIc. Species alignments were performed using Clustal W1.8 (Higgins et al. 1996). Amino acid residues are shown in white letters on black if they are identical in several sodium-phosphate cotransporter types (type IIa: human, rat, mouse, dog, chimpanzee, and chicken; type IIb: human, rat, mouse, dog, chimpanzee, and chicken; type IIc: human, rat, mouse, and dog). Amino acid residues are shown in black letters on gray if they are identical in all listed species for one sodium-phosphate cotransporter type. Amino acid residues not conserved among species for one sodium-phosphate cotransporter type are shown in black letters on white. Gray horizontal bars indicate the predicted location of membrane-spanning domains in NaP_i-IIc (see Segawa et al. 2002). Squares above the IIa sequence indicate amino acid residues identified by site-directed mutagenesis in mouse and rat NaP_i-IIa that are important for transport function, circles indicate residues important for PTH-mediated internalization, and asterisks (*) indicate cysteine residues thought to form intramolecular cysteine bonds. Black arrowheads indicate residues affected by point mutations. Two of these residues, S192 and G196, when mutated in the corresponding position of mouse Npt2c to the respective residues of mouse Npt2a, confer 3:1 sodium:phosphate stoichiometry and electrogenicity to Npt2c (Bacconi et al. 2005). See Web Resources for GenBank accession numbers of sequences.