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Impaired phosphate transport in SLC34A2 variants in patients with pulmonary alveolar microlithiasis
Åsa Lina M Jönsson1,2, Nati Hernando3,4, Thomas Knöpfel3,4
1Department of Biomedicine, Aarhus University, Aarhus, Denmark. aasajoen@rm.dk.
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 32Pi 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 32Pi 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.
Insights
Genetic variants in SLC34A2 cause pulmonary alveolar microlithiasis (PAM). This study found that while one variant allowed protein expression, all tested variants resulted in non-functional sodium-dependent phosphate transport protein 2b (NaPi-IIb), confirming its role in PAM.
Area of Science:
- Genetics
- Molecular Biology
- Pathology
Background:
- Pulmonary alveolar microlithiasis (PAM) is a rare lung disease caused by variants in the SLC34A2 gene, which encodes the sodium-dependent phosphate transport protein 2b (NaPi-IIb).
- PAM is characterized by progressive calcium-phosphate deposition in the alveoli, with no current effective treatments.
- While numerous SLC34A2 variants have been identified in patients, few have been functionally characterized.
Purpose of the Study:
- To investigate the functional impact of specific SLC34A2 variants on NaPi-IIb transporter expression and phosphate uptake.
- To correlate observed molecular defects with the pathogenesis of pulmonary alveolar microlithiasis.
Main Methods:
- Selected nonsense, frameshift, and in-frame deletion variants of SLC34A2 were engineered into human NaPi-IIb constructs.
- Constructs were expressed in Xenopus laevis oocytes, and phosphate uptake was measured using a 32Pi assay.
- NaPi-IIb protein expression and membrane localization were assessed via immunoblotting and immunohistochemistry.
Main Results:
- Wild-type NaPi-IIb demonstrated significant phosphate transport and correct membrane localization in oocytes.
- The in-frame deletion variant (Thr468del) showed protein expression and membrane localization but lacked phosphate transport function.
- All other analyzed variants, including nonsense and frameshift mutations, resulted in non-functional transporters with no detectable membrane expression.
Conclusions:
- The functional analysis of SLC34A2 variants provides direct evidence that NaPi-IIb transporter dysfunction underlies pulmonary alveolar microlithiasis.
- Only the Thr468del variant exhibited partial protein expression, but all variants were non-functional, reinforcing the critical role of NaPi-IIb in phosphate homeostasis and PAM pathogenesis.
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