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
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
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DOI：
10.1152/ajprenal.00090.2008
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发表时间：
2008-08-01
影响因子：
4.2
通讯作者：
Bergwitz, Clemens
中科院分区：
文献类型：
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作者：
Jaureguiberry, Graciana;Carpenter, Thomas O.;Bergwitz, Clemens
本研究描述了两个新的复合杂合突变，c。410C>T（p.T137M）（T137M）; 4225_50del在SLC 34 A3的父系等位基因上，在先前报道的患有遗传性低磷酸盐血症佝偻病伴高钙尿症（HHRH）和复发性肾结石的男性中（Chen C，Carpenter T，Steg N，Baron R，Anast C. Pediatrics 84：276-280，1989）。对于体外功能分析，我们产生了编码增强的绿色荧光蛋白（EGFP）的表达质粒，所述增强的绿色荧光蛋白（EGFP）连接到野生型或突变型人IIc型Na-P-1协同转运蛋白（NaPi-IIc）的NH 2末端，即，EGFP-hNaPi-IIc、EGFP-[M137] hNaPi-IIc或EGFP[Stop446] hNaPi-IIc。V446 Stop突变体在负鼠肾（OK）细胞和钠依赖性P-33摄取到非洲爪蟾卵母细胞的顶端补丁表达测定时，表现出完全丧失表达和功能。相反，将EGFP-[M137] hNaPi-IIc插入OK细胞的顶端斑块和卵母细胞膜中。然而，当通过共聚焦显微镜定量时，与野生型相比，表面荧光减少至40%。经表面表达校正后，由EGFP-[M137] hNaPi-IIc介导的卵母细胞的33 P摄取率降低了60%。该NaPi-IIc突变体的功能总体降低至16%，同时g. 4225_50del（V446 Stop），因此似乎足以解释我们患者的表型。此外，与野生型相比，EGFP[M137] hNaPi-IIc的Na-22和P-33吸收的化学计量比增加至7.1 +/- 3.65。双电极研究表明，EGFP-[M137] hNaPi-IIc是非产电的，但显示出显着的磷酸盐非依赖性内向整流钠电流，这似乎是不敏感的膦甲酸。因此，M137可能解偶联钠-磷酸盐共转运，这表明该氨基酸残基在人NaPi-IIc中具有重要的功能作用。
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-P-i 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 P-33 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 Na-22 and P-33 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.