SLC34A2 变体导致的磷酸盐转运功能障碍与肺泡微结石症相关。

Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis.

机构信息

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark.

出版信息

Hum Genomics. 2022 Apr 20;16(1):13. doi: 10.1186/s40246-022-00387-y.

DOI:10.1186/s40246-022-00387-y

PMID:35443721

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9019944/

Abstract

BACKGROUND

Variants in SLC34A2 encoding the sodium-dependent phosphate transport protein 2b (NaPi-IIb) cause the rare lung disease pulmonary alveolar microlithiasis (PAM). PAM is characterised by the deposition of calcium-phosphate concretions in the alveoli usually progressing over time. No effective treatment is available. So far, 30 allelic variants in patients have been reported but only a few have been functionally characterised. This study aimed to determine the impact of selected SLC34A2 variants on transporter expression and phosphate uptake in cellular studies.

METHODS

Two nonsense variants (c.910A > T and c.1456C > T), one frameshift (c.1328delT), and one in-frame deletion (c.1402_1404delACC) previously reported in patients with PAM were selected for investigation. Wild-type and mutant c-Myc-tagged human NaPi-IIb constructs were expressed in Xenopus laevis oocytes. The transport function was investigated with a Pi uptake assay. NaPi-IIb protein expression and localisation were determined with immunoblotting and immunohistochemistry, respectively.

RESULTS

Oocytes injected with the wild-type human NaPi-IIb construct had significant Pi transport compared to water-injected oocytes. In addition, the protein had a molecular weight as expected for the glycosylated form, and it was readily detectable in the oocyte membrane. Although the protein from the Thr468del construct was synthesised and expressed in the oocyte membrane, phosphate transport was similar to non-injected control oocytes. All other mutants were non-functional and not expressed in the membrane, consistent with the expected impact of the truncations caused by premature stop codons.

CONCLUSIONS

Of four analysed SLC34A2 variants, only the Thr468del showed similar protein expression as the wild-type cotransporter in the oocyte membrane. All mutant transporters were non-functional, supporting that dysfunction of NaPi-IIb underlies the pathology of PAM.

摘要

背景

SLC34A2 编码的钠依赖性磷酸盐转运蛋白 2b（NaPi-IIb）的变体导致罕见的肺部疾病肺泡微结石症（PAM）。PAM 的特征是肺泡中钙磷酸盐结石的沉积，通常随着时间的推移而进展。目前尚无有效的治疗方法。迄今为止，已有 30 种患者等位基因变异被报道，但仅有少数具有功能特征。本研究旨在通过细胞研究确定选定的 SLC34A2 变体对转运蛋白表达和磷酸盐摄取的影响。

方法

选择先前在 PAM 患者中报道的两个无义变异（c.910A>T 和 c.1456C>T）、一个移码变异（c.1328delT）和一个框内缺失（c.1402_1404delACC）进行研究。野生型和突变型 c-Myc 标记的人 NaPi-IIb 构建体在非洲爪蟾卵母细胞中表达。用 Pi 摄取测定法研究转运功能。通过免疫印迹和免疫组织化学分别确定 NaPi-IIb 蛋白表达和定位。

结果

与水注射卵母细胞相比，注射野生型人 NaPi-IIb 构建体的卵母细胞具有显著的 Pi 转运。此外，该蛋白的分子量与糖基化形式预期一致，并且在卵母细胞膜中易于检测。尽管 Thr468del 构建体的蛋白在卵母细胞膜中合成和表达，但磷酸盐转运与非注射对照卵母细胞相似。所有其他突变体均无功能，不能在膜中表达，这与提前终止密码子引起的截短预期影响一致。