Abstract
Primary familial brain calcification (PFBC) is a neurological disease characterized by calcium phosphate deposits in the basal ganglia and other brain regions and has thus far been associated with SLC20A2, PDGFB or PDGFRB mutations. We identified in multiple families with PFBC mutations in XPR1, a gene encoding a retroviral receptor with phosphate export function. These mutations alter phosphate export, implicating XPR1 and phosphate homeostasis in PFBC.
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Acknowledgements
We acknowledge and thank all of the participants and families for their valuable contributions to our study; our clinical staff and laboratory members, J. DeYoung and the University of California Los Angeles (UCLA) Neuroscience Genomics Core, J. Touhami and J. Laval for their assistance and constant support; and the National Heart, Lung, and Blood Institute (NHLBI) GO Exome Sequencing Project and its ongoing studies, which produced and provided exome variant calls for comparison: the Lung GO Sequencing Project (HL-102923), the Women's Health Initiative (WHI) Sequencing Project (HL-102924), the Broad GO Sequencing Project (HL-102925), the Seattle GO Sequencing Project (HL-102926) and the Heart GO Sequencing Project (HL-103010). We are also indebted to the Montpellier Rio Imaging (MRI) platform for flow cytometry experiments. This work was funded by the US National Institutes of Health/National Institute of Neurological Disorders and Stroke (R01NS040752 to D.H.G.), by Association Française contre les Myopathies (AFM) and Ligue Nationale contre le Cancer (Comité de l'Hérault; to J.-L.B.), and by Fondation pour la Recherche Médicale (FRM) and a FEDER European Union Languedoc-Roussillon grant (Transportome; to M.S.). We also acknowledge the support of the National Institute of Neurological Disorders and Stroke Informatics Center for Neurogenetics and Neurogenomics (PSNS062691). D.G. was supported by FRM, Institut National du Cancer (INCa) and Labex GR-Ex (ANR-11-LABX-0051) fellowships, and U.L.-S. was supported by a Labex EpiGenMed (ANR-10-LABX-12-01) fellowship; Labex is funded by the program 'Investissements d'Avenir' of the French National Research Agency. J.-L.B. and M.S. were supported by INSERM. M.-J.S. and B.Q. are supported by the Fondo de Investigación Sanitaria, grant PI12/00742; INNOPHARMA project MINECO-USC; and FEDER funds. M.-J.S. and B.Q. hold research contracts from the Institute of Health Carlos III–SERGAS. J.R.M.O. acknowledges funding from FACEPE (APQ 1831-4.01/12) and CNPq (457556/2013-7; 480255/2013-0; 307909/2012-3). B.L.F. is funded by US National Institutes of Health grants K08MH086297 (National Institute of Mental Health) and R01NS082094 (National Institute of Neurological Disorders and Stroke). G.N., A.-C.R., D.H. and D.C. are supported by INSERM, the University Hospital of Rouen and the French CNR-MAJ.
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M.S., D.H.G., J.-L.B. and G.C. designed the study. A.L., D.G., G.N. and U.L.-S. designed and performed experiments. A.L., D.G., G.N., U.L.-S., B.Q., J.R.M.O., L.S., E.M.R., E.S., M.-J.S., A.-C.R., D.C., M.S., D.H.G., J.-L.B. and G.C. analyzed data. A.L., D.G., M.S., J.-L.B. and G.C. wrote the manuscript. A.L., D.G., G.N., J.R.M.O., R.L.S., E.M.R., M.-J.S., B.L.F., A.E.L., Z.M., H.P., P. Striano, V.K.U., M.W.W., M.S., J.-L.B. and G.C. edited the manuscript. G.N., B.Q., J.R.M.O., E.S., M.-J.S., Á.C., C.C.-F., S.C., B.L.F., C.G., J.C.J., S.K., A.E.L., Z.M., W.M., M.P., H.P., J.P., N.S.S., S.A.S., P. Striano, P. Svenningsson, F.T., V.K.U., O.V., M.W.W., S.W., M.Y., F.B., D.H., D.H.G. and G.C. recruited and evaluated patients and collected blood samples.
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J.-L.B. and M.S. are inventors on a provisional patent describing the use of ligands, including XRBD, for the analysis of human cells (PCT/EP2010/050139), and M.S. is a co-founder of METAFORA-biosystems, a start-up company that focuses on metabolite transporters under physiological and pathological conditions.
Integrated supplementary information
Supplementary Figure 1 Exome sequencing in five members of the PFBC family and identification of four candidate variants.
(a) Pedigree of the PFBC family. Shown are unaffected (open), affected (filled) and uncertain clinical state (gray) family members with homozygote reference sequence (o) and heterozygote mutation (*). (b) Filtering of the variants identified by exome sequencing.
Supplementary Figure 2 Reduced pedigrees of French families with XPR1 variant carriers.
Affected individuals with pathological calcification, as visualized by computed tomography (CT) scan, are represented by solid symbols; open symbols represent individuals with CT scans showing no pathological calcification and gray symbols are used for individuals with unknown status (no available CT scan). With the exception of the parents of patient ROU-1025-001, only informative relatives are shown. When available, characteristic axial views of CT scans of patients and unaffected relatives are included next to the corresponding individual. White arrows indicate areas of brain calcification.
Supplementary Figure 3 XPR1 p.Leu145Pro alteration has no effect on phosphate uptake.
(a) Uptake of 33P in HEK293T cells transfected as in Figure 1. Results are means ± s.e.m. from a representative experiment (n = 3). (b) Cell surface expression of PiT1 (top) and PiT2 (bottom) as monitored by flow cytometry. Numbers correspond to the delta mean fluorescence values. Shown is a representative experiment (n = 3).
Supplementary Figure 4 Predicted damaging XPR1 mutants and not the p.Lys53Arg non-damaging mutant alter inorganic phosphate efflux.
(a) 33P efflux in HEK293T cells transfected with siLUC (lane 1), siXPR1 alone (lane 2) or siXPR1 in combination with HA-tagged XPR1 expression vectors harboring either wild-type XPR1 (lane 3), the non-damaging p.Lys53Arg mutant (lane 4) or the damaging p.Ser136Asn (lane 5), p.Leu140Pro (lane 6) or p.Leu218Ser (lane 7) XPR1 mutants. Shown are means ± s.e.m. (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (b) XPR1 (top), PiT2 (middle) and PiT1 (bottom) cell surface expression as monitored by flow cytometry using XRBD, AmphoRBD and KoRBD, respectively. Numbers represent the specific mean fluorescence intensity of one representative of three independent experiments.
Supplementary Figure 5 Multiple-sequence alignment of orthologous XPR1 proteins from different species.
Multiple alignment of the amino acid sequence of human XPR1 (NP_004727.2) with the orthologous proteins of Felis catus (XP_011289290.1), Rattus norvegicus (NP_001099462.1), Mus musculus (NP_035403.1), Didelphis virginiana (AHI85562.1), Xenopus laevis (NP_001086930.1), Danio rerio (NP_001232029.1) and Drosophila melanogaster (XP_001977625.1) performed with ClustalW2. Residues that differ from human XPR1 are shown in gray, and different colors indicate hydrophobic (AFILMVW; blue), basic (KR; red), acidic (DE; magenta), or large and aromatic (HY; light blue) residues, while disulfide bonding (C; salmon), flexible (G; orange) and rigid (P; yellow) residues are singled out and other polar residues are grouped (NQST; green). The positions of residues altered by mutations identified in this study are boxed.
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Legati, A., Giovannini, D., Nicolas, G. et al. Mutations in XPR1 cause primary familial brain calcification associated with altered phosphate export. Nat Genet 47, 579–581 (2015). https://doi.org/10.1038/ng.3289
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DOI: https://doi.org/10.1038/ng.3289
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