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1 Endocrine Unit, Massachusetts General Hospital, Boston, Massachusetts, United States
2 Department of Pediatrics, Endocrinology, Yale University School of Medicine, New Haven,, Connecticut, United States
3 Dept. of Anesthesiology, Massachusetts General Hospital, Boston, Massachusetts, United States
* To whom correspondence should be addressed. E-mail: cbergwitz{at}partners.org.
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 (Pediatrics 1989; 84:276-280). For functional analysis in vitro, we generated expression plasmids encoding enhanced green-fluorescence protein (EGFP) concatenated to the N-terminus of wild-type or mutant human 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 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 to 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 when compared to wild-type. Two-electrode studies indicate that EGFP-[M137]hNaPi-IIc is non-electrogenic, but displayed a significant phosphate-independent inward-rectified sodium-current, which appears to be insensitive to phosphonoformic acid. M137 thus may uncouple sodium-phosphate co-transport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.
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