Hereditary hypophosphatemic rickets with hypercalciuria is caused by mutations in the sodium-phosphate cotransporter gene SLC34A3

Abstract

Hypophosphatemia due to isolated renal phosphate wasting results from a heterogeneous group of disorders. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive form that is characterized by reduced renal phosphate reabsorption, hypophosphatemia, and rickets. It can be distinguished from other forms of hypophosphatemia by increased serum levels of 1,25-dihydroxyvitamin D resulting in hypercalciuria. Using SNP array genotyping, we mapped the disease locus in two consanguineous families to the end of the long arm of chromosome 9. The candidate region contained a sodium-phosphate cotransporter gene, SLC34A3, which has been shown to be expressed in proximal tubulus cells. Sequencing of this gene revealed disease-associated mutations in five families, including two frameshift and one splice-site mutation. Loss of function of the SLC34A3 protein presumably results in a primary renal tubular defect and is compatible with the HHRH phenotype. We also show that the phosphaturic factor FGF23 (fibroblast growth factor 23), which is increased in X-linked hypophosphatemic rickets and carries activating mutations in autosomal dominant hypophosphatemic rickets, is at normal or low-normal serum levels in the patients with HHRH, further supporting a primary renal defect. Identification of the gene mutated in a further form of hypophosphatemia adds to the understanding of phosphate homeostasis and may help to elucidate the interaction of the proteins involved in this pathway.

Figure 1

Mutation analysis of SLC34A3 in five families (left) and electropherograms of the affected individuals (right). The mutations are indicated above the electropherograms. Families 1 and 2 presented homozygous mutations. Affected individuals from families 3 and 4 were compound heterozygous. In the affected individual from family 5, we found only a heterozygous deletion. Affected individuals are indicated by fully blackened symbols, and unaffected heterozygous individuals by half-blackened symbols.

Figure 2

Multipoint linkage analysis of 15 cM of the telomeric end of the long arm of chromosome 9. The analyses were performed using ALLEGRO (q=0.001; f₁=0.0; f₂=0.0; f₃=0.99) and included affected and unaffected family members. Depicted are summary LOD scores under the assumption of homogeneity. Analysis of SNP markers was performed in families (Fam) 1 and 2 (black line), and analyses of microsatellite markers were performed in all families, once with (bottom gray line) and once without (top gray line) consanguinity in family 3. The SLC34A3 gene maps between D9S905 and D9S1838.

Figure 3

Genomewide linkage analysis of families 1 and 2. Analysis was performed using ALLEGRO (q=0.001; f₁=0.0; f₂=0.0; f₃=0.99) and included affected and unaffected family members. Depicted are summary LOD scores under the assumption of homogeneity.

Figure 4

Amino acid alignment of SLC34A1, SLC34A2, and SLC34A3 proteins from different vertebrate species. The proteins were obtained from GenBank, the UCSC Genome Browser, or Ensembl or were predicted by tblastn and Genewise from the corresponding genome sequence. Part of the predicted sequences is supplemented with partial cDNA sequences. The alignment was generated using ClustalW and Prettybox. Polymorphisms and variations of uncertain effect are indicated by plus signs (+), and missense mutations are indicated by asterisks (*). GenBank sequences (and accession numbers) include Human_a1 (NP_003043.2), Human_a2 (NP_006415.1), Human_a3 (NP_543153.1), Mouse_a1 (NP_035522.1), Mouse_a2 (NP_035532.1), Mouse_a3 (NP_543130.1), Chicken_a2 (NP_989805), Xenopus_a2 (AAH67316.1), Zebrafish_a2a (NP_571699.1), and Zebrafish_a2b (AAG35356.2). Ensembl proteins (and accession numbers) include Chicken_a1 (ENSGALP00000004850 and BU122185), Xenopus_a3 (ENSXETP00000050672 and CX972852), Zebrafish_a1a (ENSDARP017391 and C0920054), Zebrafish_a1b (ENSDARP00000041987 and CR929642), Fugu_a1 (SINFRUP00000132898), and Fugu_a2 (SINFRUP00000149494). Predicted proteins include Opossum_a1, Opossum_a2a, Opossum_a2b, Opossum_a2c, Opossum_a3, and Tetraodon_a2b.

Figure 4

Amino acid alignment of SLC34A1, SLC34A2, and SLC34A3 proteins from different vertebrate species. The proteins were obtained from GenBank, the UCSC Genome Browser, or Ensembl or were predicted by tblastn and Genewise from the corresponding genome sequence. Part of the predicted sequences is supplemented with partial cDNA sequences. The alignment was generated using ClustalW and Prettybox. Polymorphisms and variations of uncertain effect are indicated by plus signs (+), and missense mutations are indicated by asterisks (*). GenBank sequences (and accession numbers) include Human_a1 (NP_003043.2), Human_a2 (NP_006415.1), Human_a3 (NP_543153.1), Mouse_a1 (NP_035522.1), Mouse_a2 (NP_035532.1), Mouse_a3 (NP_543130.1), Chicken_a2 (NP_989805), Xenopus_a2 (AAH67316.1), Zebrafish_a2a (NP_571699.1), and Zebrafish_a2b (AAG35356.2). Ensembl proteins (and accession numbers) include Chicken_a1 (ENSGALP00000004850 and BU122185), Xenopus_a3 (ENSXETP00000050672 and CX972852), Zebrafish_a1a (ENSDARP017391 and C0920054), Zebrafish_a1b (ENSDARP00000041987 and CR929642), Fugu_a1 (SINFRUP00000132898), and Fugu_a2 (SINFRUP00000149494). Predicted proteins include Opossum_a1, Opossum_a2a, Opossum_a2b, Opossum_a2c, Opossum_a3, and Tetraodon_a2b.

Figure 4

Amino acid alignment of SLC34A1, SLC34A2, and SLC34A3 proteins from different vertebrate species. The proteins were obtained from GenBank, the UCSC Genome Browser, or Ensembl or were predicted by tblastn and Genewise from the corresponding genome sequence. Part of the predicted sequences is supplemented with partial cDNA sequences. The alignment was generated using ClustalW and Prettybox. Polymorphisms and variations of uncertain effect are indicated by plus signs (+), and missense mutations are indicated by asterisks (*). GenBank sequences (and accession numbers) include Human_a1 (NP_003043.2), Human_a2 (NP_006415.1), Human_a3 (NP_543153.1), Mouse_a1 (NP_035522.1), Mouse_a2 (NP_035532.1), Mouse_a3 (NP_543130.1), Chicken_a2 (NP_989805), Xenopus_a2 (AAH67316.1), Zebrafish_a2a (NP_571699.1), and Zebrafish_a2b (AAG35356.2). Ensembl proteins (and accession numbers) include Chicken_a1 (ENSGALP00000004850 and BU122185), Xenopus_a3 (ENSXETP00000050672 and CX972852), Zebrafish_a1a (ENSDARP017391 and C0920054), Zebrafish_a1b (ENSDARP00000041987 and CR929642), Fugu_a1 (SINFRUP00000132898), and Fugu_a2 (SINFRUP00000149494). Predicted proteins include Opossum_a1, Opossum_a2a, Opossum_a2b, Opossum_a2c, Opossum_a3, and Tetraodon_a2b.

Figure 5

Dendrogram of SLC34A1, SLC34A2, and SLC34A3 proteins from different vertebrate species. The sequences were obtained from GenBank or Ensembl or were predicted by tblastn and Genewise from the corresponding genome sequence and were aligned using ClustalW (fig. 4).