title: A novel missense mutation in SLC34A3 that causes hereditary hypophosphatemic rickets with hypercalciuria in humans identifies threonine 137 as an important determinant of sodium-phosphate cotransport in NaPi-IIc.
abstract: The present study describes two novel compound heterozygous mutations, c.410C>T(p.T137M) (T137M) on the maternal and g.4225_50del on the paternal allele of SLC34A3, in a previously reported male with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and recurrent kidney stones (Chen C, Carpenter T, Steg N, Baron R, Anast C. Pediatrics 84: 276-280, 1989). For functional analysis in vitro, we generated expression plasmids encoding enhanced green fluorescence protein (EGFP) concatenated to the NH2 terminus of wild-type or mutant human type IIc Na-Pi cotransporter (NaPi-IIc), i.e., EGFP-hNaPi-IIc, EGFP-[M137]hNaPi-IIc, or EGFP-[Stop446]hNaPi-IIc. The V446Stop mutant showed complete loss of expression and function when assayed for apical patch expression in opossum kidney (OK) cells and sodium-dependent 33P uptake into Xenopus laevis oocytes. Conversely, EGFP-[M137]hNaPi-IIc was inserted into apical patches of OK cells and into oocyte membranes. However, when quantified by confocal microscopy, surface fluorescence was reduced to 40% compared with wild-type. After correction for surface expression, the rate of 33P uptake by oocytes mediated by EGFP-[M137]hNaPi-IIc was decreased by an additional 60%. The resulting overall reduction of function of this NaPi-IIc mutant to 16%, taken together with complete loss of expression and function of g.4225_50del(V446Stop), thus appears to be sufficient to explain the phenotype in our patient. Furthermore, the stoichiometric ratio of 22Na and 33P uptake was increased to 7.1 +/- 3.65 for EGFP-[M137]hNaPi-IIc compared with wild-type. Two-electrode studies indicate that EGFP-[M137]hNaPi-IIc is nonelectrogenic but displayed a significant phosphate-independent inward-rectified sodium current, which appears to be insensitive to phosphonoformic acid. M137 thus may uncouple sodium-phosphate cotransport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.
authors.affiliation: Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts, USA.
authors.forename: Graciana
authors.initials: G
authors.lastname: Jaureguiberry
authors.forename: Thomas O
authors.initials: TO
authors.lastname: Carpenter
authors.forename: Stuart
authors.initials: S
authors.lastname: Forman
authors.forename: Harald
authors.initials: H
authors.lastname: Jüppner
authors.forename: Clemens
authors.initials: C
authors.lastname: Bergwitz
mesh_terms: D000328:Adult; D000483:Alleles; D000818:Animals; D001483:Base Sequence; D005089:Exocytosis / Q000502:physiology; D053098:Familial Hypophosphatemic Rickets / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006239:Haplotypes / Q000235:genetics; D006801:Humans; D053565:Hypercalciuria / Q000235:genetics* / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics*; D009865:Oocytes / Q000166:cytology / Q000378:metabolism; D009893:Opossums; D010710:Phosphates / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D050608:Sodium-Phosphate Cotransporter Proteins / Q000378:metabolism*; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc / Q000235:genetics* / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D010710:Phosphates; C493562:SLC34A3 protein, human; D050608:Sodium-Phosphate Cotransporter Proteins; D050615:Sodium-Phosphate Cotransporter Proteins, Type IIc; D013912:Threonine; D012964:Sodium