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J Mol Neurosci
2017 Feb 01;612:215-220. doi: 10.1007/s12031-016-0868-7.
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Primary Brain Calcification Causal PiT2 Transport-Knockout Variants can Exert Dominant Negative Effects on Wild-Type PiT2 Transport Function in Mammalian Cells.
Larsen FT
,
Jensen N
,
Autzen JK
,
Kongsfelt IB
,
Pedersen L
.
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Primary brain calcification (PBC) is a neurodegenerative disorder characterized by calcium-phosphate deposits in the basal ganglia and often also other areas of the brain. The prevalent clinical manifestations are cognitive impairment, neuropsychiatric symptoms, and movement disorders. In recent years, monoallelic variants in SLC20A2, which encodes the type III sodium-dependent inorganic phosphate (Pi) transporter 2 (PiT2), have been linked to the familial form of PBC in 40-50% of the families reported worldwide as well as to sporadic cases of PBC. Further insight into the disease mechanism is, however, needed. Based on co-expression studies of wild-type and variant PiT2 in Xenopus laevis oocytes, the molecular disease mechanism associated with SLC20A2 missense variants has formerly been suggested to be haploinsufficiency. We have here used mammalian cells isolated from a Slc20a2 (-/-) mouse and co-expression of human wild-type and variant PiT2. Two of the variants studied have both been reported twice in unrelated PBC cases: PiT2D28N in two sporadic cases and PiT2E575K in a familial and a sporadic case. We find that in mammalian cells, the analyzed SLC20A2 missense variants can exert their effect in a dominant negative manner resulting in decreased wild-type PiT2 Pi transport. Thus, compared to monoallelic lack of functional PiT2 protein expression, which reasonably points towards haploinsufficiency, certain SLC20A2 missense variants may be more detrimental for cellular Pi uptake and potentially contribute to an earlier disease onset and/or a more severe phenotype as observed for Slc20a2 (-/-) mice compared to Slc20a2 (+/-) mice.
Fig. 1. Co-expression assays. Cells were seeded at 1.5 × 104 cells/cm2 in four-well plates. The following day, they were transfected with WT PiT2-encoding vector, pOJ74, and vectors encoding the Pi-transport-incompetent a PiT2E575K (pOJ74E575K) or b PiT2D28N (pOJ74D28N) or PiT2H502A (pOJ74H502A) as indicated. Mock-transfected: cells transfected with empty vector DNA. Ratios refer to the ratio between vectors encoding WT and Pi-transport-incompetent PiT2, where 1 represents 0.05 μg DNA. Emtpy expression vector and plasmid DNAs were included to ensure equimolar amounts of expression vector and same amount of DNA, respectively, in the transfections. Approximately 48 h after transfection, the cells were incubated in transport medium containing a total [Pi] of 1 mM and the import of 32Pi was analyzed over 5 min. Results are shown as mean Pi uptake per microgram protein per hour of at least three wells; error bars represent SDs. Asterisk indicates p ≤ 0.05 relative to cells expressing WT PiT2 only (pOJ74 (1:0) (leftmost column))
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