The Role of Human HHRH Mutations in the Function of NaPi-IIc

人类 HHRH 突变在 NaPi-IIc 功能中的作用

• 批准号: 8116538
• 负责人: Clemens Bergwitz
• 金额: $ 8.53万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2012-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8116538
• 关键词: Address; Affect; Age; Animals; Blood; Cell Line; Cells; Co-Immunoprecipitations; Data; Deletion Mutation; Dimerization; Disease; Dominant-Negative Mutation; Evaluation; Familial hypophosphatemic bone disease; Fluorescence Resonance Energy Transfer; Homeostasis; Human; Hypercalcemia; Hypophosphatemia; In Vitro; Individual; Inherited; Kidney; Kidney Calculi; Knock-in Mouse; Lead; Life; Mammalian Cell; Metabolic; Methods; Missense Mutation; Mus; Mutation; Nephrocalcinosis; Patients; Phenotype; Relative (related person); Role; Techniques; Technology; Testing; Transgenic Organisms; Tubular formation; Urine; Xenopus oocyte; calcification; crosslink; dimer; hypercalciuria; in vivo; inorganic phosphate; mouse model; mutant; protein aggregation; public health relevance; research study; sodium phosphate; symporter; ward

DESCRIPTION (provided by applicant): This project is going to address the question whether the sodium-phosphate co-transporter NaPi-IIc forms dimers or multimers when expressed in Xenopus oocytes and mammalian cell lines and whether human mutations in NaPi-IIc interfere with sodium-phosphate co-transport by the formation of non-functional heterodimers. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by homozygous or compound heterozygous missense or deletion mutations in SLC34A3/NaPi-IIc. HHRH is a disorder that persists life-long and heterozygous relatives of individuals affected by HHRH often have hypercalciuria and renal calcifications due to elevated 1,25(OH)2D levels as a result of renal phosphate losses. In contrast, mice lacking SLC34A3/NaPi-IIc show only mild hypercalcemia and absorptive hypercalciuria at an early age due to increased 1,25(OH)2D levels, but these animals do not develop hypophosphatemia, and heterozygous animals are normal. While haploinsufficiency could explain the hypercalciuria observed in heterozygous human carriers of NaPi-IIc mutations, it is therefore possible that some mutations, particularly those associated with a more severe phenotype such as renal stones, lead to dominant negative effects on wild-type NaPi-IIc or other sodium-phosphate co-transporters. In the past two years since the start of my KO8 "Role of the renal sodium-phosphate co-transporter NaPi-IIc in phosphate homeostasis" I have established all techniques required for functional analysis of SLC34A3/NaPi-IIc mutations, which were discovered in patients referred with hereditary hypophosphatemic rickets with hypercalciuria (HHRH) and related disorders. When co-expressed in Xenopus oocytes, the mutants T137M and V446Stop impair wild-type NaPi-IIc function in a dominant-negative fashion, while NaPi-IIa function was unaffected. Using cross-linking and co-immunoprecipitation techniques, I have shown dimerization of wild-type NaPi-IIc after over-expression in Xenopus oocytes. These experiments were carefully controlled for non-specific protein aggregation effects. My plan is to repeat these studies in OK, HEK293 and LLCPK-1 cells to show that dimerization also occurs in mammalian proximal tubular cell lines. I also plan to establish the FRET technology, which does not require cross-linking and purification of dimers as an independent method to show that dimerization occurs. I will furthermore use adenoviral transduction of mutant and wild-type NaPi-IIc to show the dominant negative effect in OK, HEK293 and LLCPK-1 cells. The proposed in vitro studies will provide first proof that dimerization of NaPi-IIc is important for it's function. If successful, these data will support a systematic evaluation of the human heterozygous carriers of T137M and V446Stop in metabolic ward studies, and the use of transgenic or knock-in mouse models to test the hypothesis whether these mutations can have a dominant negative effect and lead to kidney stone formation and/or nephrocalcinosis in vivo. PUBLIC HEALTH RELEVANCE: SLC34A3/NaPi-IIc is a renal sodium-phosphate co-transporter, which helps reclaiming phosphate from the urine to increase blood phosphate levels. Homozygous mutations in NaPi-IIc cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH) in humans, but it is unclear why also heterozygous carriers of NaPi-IIc mutations sometimes have a relatively severe phenotype such as renal stones. This proposal is going to test the hypothesis that some mutations may interfere with wild-type co-transporters by the formation of non-functional heterodimers thereby explaining the relatively severe phenotype of heterozygous carriers.

描述（由申请人提供）：本项目将解决钠-磷酸盐共转运蛋白NaPi-IIc在非洲爪蟾卵母细胞和哺乳动物细胞系中表达时是否形成二聚体或多聚体以及NaPi-IIc的人类突变是否通过形成非功能性异源二聚体干扰钠-磷酸盐共转运的问题。 遗传性低磷血症性佝偻病伴高钙尿症（HHRH）是由SLC 34 A3/NaPi-IIc基因纯合或复合杂合错义或缺失突变引起的。HHRH是一种终生持续的疾病，受HHRH影响的个体的杂合子亲属通常由于肾磷酸盐损失导致的1，25（OH）2D水平升高而具有高钙尿症和肾钙化。相比之下，缺乏SLC 34 A3/NaPi-IIc的小鼠由于1，25（OH）2D水平增加而在早期仅显示轻度高钙血症和吸收性高钙尿症，但这些动物不发生低磷酸盐血症，并且杂合动物是正常的。虽然单倍不足可以解释在NaPi-IIc突变的杂合子人类携带者中观察到的高钙尿症，但因此可能是某些突变，特别是与更严重表型如肾结石相关的突变，导致对野生型NaPi-IIc或其他磷酸钠共转运蛋白的显性负效应。在过去的两年里，自从我的KO 8“肾钠磷酸盐协同转运蛋白NaPi-IIc在磷酸盐稳态中的作用”开始以来，我已经建立了SLC 34 A3/NaPi-IIc突变的功能分析所需的所有技术，这些突变在遗传性低磷酸盐血症佝偻病伴高钙尿症（HHRH）和相关疾病的患者中发现。当在非洲爪蟾卵母细胞中共表达时，突变体T137 M和V446 Stop以显性负性方式损害野生型NaPi-IIc功能，而NaPi-IIa功能不受影响。使用交联和免疫共沉淀技术，我已经显示了野生型NaPi-IIc在非洲爪蟾卵母细胞中过度表达后的二聚化。对这些实验进行仔细控制，以获得非特异性蛋白质聚集效应。我的计划是在OK，HEK 293和LLCPK-1细胞中重复这些研究，以表明二聚化也发生在哺乳动物近端肾小管细胞系中。我还计划建立FRET技术，该技术不需要交联和纯化二聚体，作为一种独立的方法来显示二聚化的发生。我将进一步使用突变型和野生型NaPi-IIc的腺病毒转导来显示OK、HEK 293和LLCPK-1细胞中的显性负效应。拟议的体外研究将首次证明NaPi-IIc的二聚化对其功能很重要。如果成功，这些数据将支持在代谢病房研究中对T137 M和V446 Stop的人类杂合携带者进行系统评价，以及使用转基因或敲入小鼠模型来测试这些突变是否具有显性负效应并导致体内肾结石形成和/或肾钙质沉着症的假设。 公共卫生相关性：SLC 34 A3/NaPi-IIc是一种肾脏钠-磷酸盐协同转运蛋白，有助于从尿液中回收磷酸盐，以增加血磷水平。NaPi-IIc的纯合突变导致人类遗传性低磷酸盐血症佝偻病伴高钙尿症（HHRH），但目前尚不清楚为什么NaPi-IIc突变的杂合携带者有时也具有相对严重的表型，如肾结石。该提议将检验以下假设：某些突变可能通过形成无功能的异二聚体干扰野生型共转运蛋白，从而解释杂合携带者的相对严重的表型。