Mutations in the gene encoding PDGF-B cause brain calcifications in humans and mice

Journal name:
Nature Genetics
Volume:
45,
Pages:
1077–1082
Year published:
DOI:
doi:10.1038/ng.2723
Received
Accepted
Published online

Calcifications in the basal ganglia are a common incidental finding and are sometimes inherited as an autosomal dominant trait (idiopathic basal ganglia calcification (IBGC)). Recently, mutations in the PDGFRB gene coding for the platelet-derived growth factor receptor β (PDGF-Rβ) were linked to IBGC. Here we identify six families of different ancestry with nonsense and missense mutations in the gene encoding PDGF-B, the main ligand for PDGF-Rβ. We also show that mice carrying hypomorphic Pdgfb alleles develop brain calcifications that show age-related expansion. The occurrence of these calcium depositions depends on the loss of endothelial PDGF-B and correlates with the degree of pericyte and blood-brain barrier deficiency. Thus, our data present a clear link between Pdgfb mutations and brain calcifications in mice, as well as between PDGFB mutations and IBGC in humans.

At a glance

Figures

  1. PDGFB mutations in IBGC.
    Figure 1: PDGFB mutations in IBGC.

    (a) Pedigrees of six families with IBGC with PDGFB mutations. Numbers below the symbols indicate individuals in whom CT scans were performed. Filled symbols, individuals affected with brain calcification, including both symptomatic and asymptomatic individuals; symbols with slashes, deceased individuals; symbols with question marks, individuals probably affected as inferred by history; +, mutation carriers; −, non-carriers; *, individuals in whom whole genomes or exomes were sequenced. (b) Schematic of the PDGFB gene with the positions of the IBGC-associated mutations indicated by arrows. The region coding for the mature form of the protein is highlighted in blue, and the regions coding for the signal peptide and propeptides are shown in red and yellow, respectively. The putative extended part of the nascent protein predicted to be translated from the c.726G>C allele is represented by a dotted box. Mutations predicted to severely impair the structure of the protein are shown above, and missense changes are shown below.

  2. Calcified inclusions in the brains of Pdgfbret/ret mice.
    Figure 2: Calcified inclusions in the brains of Pdgfbret/ret mice.

    Sections of the midbrain of 2- and 4-month-old Pdgfbret/ret mice stained with hematoxylin and eosin show spheroid inclusions positive for staining with alcian blue and PAS, which are absent in littermate controls. In 4-month-old animals, these inclusions develop positivity for staining with alizarin red. Images are representative of analyses made on three mice per genotype. Scale bars, 100 μm (10 μm in insets). Arrowheads indicate the lesion shown in the inset at ×10 higher magnification.

  3. Progressive brain calcification in 1-year-old Pdgfbret/ret mice.
    Figure 3: Progressive brain calcification in 1-year-old Pdgfbret/ret mice.

    (a) Sagittal brain sections of a 1-year-old Pdgfbret/ret mouse show calcified nodules in four distinct anatomical regions: basal forebrain (BF), thalamus (T), midbrain (MB) and pons (P) that are absent in the littermate control. A single calcospheroid can be seen in the medulla. Scale bars, 1 mm (100 μm in insets). Images are representative of analyses made on four mice per genotype. (b) Micro-CT scan of the brain of a 1-year-old Pdgfbret/ret mouse. Coronal (left), sagittal (upper right) and axial (lower right) maximum-intensity projections are shown. The contrast of soft-tissue structures was increased by previous incubation in iodinated contrast agent. Note the bilaterally visible punctiform bone-dense structures within the midbrain-thalamus region (arrows). (c) Axial views of T1-weighted manganese-enhanced MRI scans of a 1-year-old Pdgfbret/ret mouse and a littermate control. Images are representative of analyses performed on three mice per genotype. Postmortem analysis of each individual verified that hypointense areas corresponded with calcifications visualized by the histostains shown in a.

  4. Elemental analysis of calcified nodules in mouse brain.
    Figure 4: Elemental analysis of calcified nodules in mouse brain.

    The laminated nodules of a 1-year-old Pdgfbret/ret mouse were examined with scanning electron microscopy combined with energy-dispersive X-ray spectroscopy. Two blocks of fixed tissue and several nodules on the same block were analyzed. (a) Scanning electron microscopy image of a lesion with laminated nodules. A nodule is located at the center of the image. Black and red plus signs indicate areas where spectral analysis was performed; the red plus sign is located in the middle of the nodule, and the black plus sign is located in the neighboring intact parenchyma. Scale bar, 10 μm. (b) Energy-dispersive X-ray spectroscopy spectra from the nodule (red line) and neighboring parenchyma (black line) shows that the nodule contains phosphorous and calcium, indicative of calcium phosphate being a primary constituent. Small peaks seen in the spectra obtained from brain parenchyma at 1.91 kV and 3.16 kV correspond with osmium and uranium, respectively, and are derived from the fixatives (black line).

  5. Brain pathology of Pdgfb-/-; R26P+/0 mice.
    Figure 5: Brain pathology of Pdgfb−/−; R26P+/0 mice.

    (a) Laminated nodules in the thalamus of 1-year-old Pdgfb−/−; R26P+/0 mice visualized by staining with hematoxylin and eosin, alcian blue and PAS have developed positivity for staining with alizarin red. Calcified nodules are absent in littermates (control and Pdgfb−/−; R26P+/+ mice). Images are representative of analyses performed on four mice per genotype. (b) A laminated nodule in the thalamus of a 5-month-old Pdgfb−/−; R26P+/0 mouse visualized by staining with hematoxylin and eosin, alcian blue and PAS is negative for staining with alizarin red. These structures are absent in the littermate control. Images are representative of analyses performed on five mice per genotype. Note the close proximity of the laminated nodule to the microvessel (inset, hematoxylin and eosin staining). Scale bars, 100 μm (10 μm in insets).

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

NCBI Reference Sequence

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Author information

  1. These authors jointly directed and contributed equally to this work.

    • Annika Keller,
    • Ana Westenberger,
    • Maria J Sobrido,
    • Christer Betsholtz,
    • Christine Klein &
    • Joao R M Oliveira

Affiliations

  1. Institute for Neuropathology, University Hospital Zürich, Zürich, Switzerland.

    • Annika Keller,
    • Elisabeth J Rushing,
    • Michael Hugelshofer,
    • Regina Reimann,
    • Irina Abakumova &
    • Adriano Aguzzi
  2. Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden.

    • Annika Keller,
    • Maarja Andaloussi Mäe,
    • Elisabeth Raschperger &
    • Christer Betsholtz
  3. Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

    • Annika Keller,
    • Maarja Andaloussi Mäe,
    • Elisabeth Raschperger &
    • Christer Betsholtz
  4. Institute of Neurogenetics, University of Lübeck, Lübeck, Germany.

    • Ana Westenberger,
    • Aloysius Domingo,
    • Katja Lohmann,
    • Katja Zschiedrich &
    • Christine Klein
  5. Fundación Pública Galega de Medicina Xenómica, Servizo Galego de Saúde (SERGAS), Instituto de Investigaciones Sanitarias (IDIS), Clinical University Hospital, Santiago de Compostela, Spain.

    • Maria J Sobrido,
    • Maria García-Murias,
    • Andres Ordoñez-Ugalde &
    • Angel Carracedo
  6. Grupo de Medicina Xenómica, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

    • Maria J Sobrido,
    • Maria García-Murias,
    • Andres Ordoñez-Ugalde &
    • Angel Carracedo
  7. Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

    • Renee L Sears,
    • Elizabeth Spiteri &
    • Daniel H Geschwind
  8. Keizo Asami Laboratory, Federal University of Pernambuco, Recife, Brazil.

    • Roberta R Lemos,
    • José E Gomes da Cunha &
    • Joao R M Oliveira
  9. Institut National de la Santé et de la Recherche Médicla (INSERM) U1079, Institute for Research and Innovation in Biomedicine (IRIB), University Hospital and Faculty of Medicine, Rouen, France.

    • Gael Nicolas,
    • Didier Hannequin &
    • Dominique Campion
  10. Department of Neurology, Rouen University Hospital, Rouen, France.

    • Gael Nicolas &
    • Didier Hannequin
  11. Centre National de Référence pour les Malades Alzheimer Jeunes (CNR-MAJ), Rouen University Hospital, Lille University Hospital and Paris-Salpêtrière University Hospital, Rouen, France.

    • Gael Nicolas,
    • Didier Hannequin &
    • Dominique Campion
  12. Center for Minimally Invasive and Endoscopic Neurosurgery, Clinic Hirslanden, Zürich, Switzerland.

    • Michael Hugelshofer
  13. Department of Diagnostic and Interventional Radiology, University Hospital Zürich, Zürich, Switzerland.

    • Moritz C Wurnig &
    • Andreas Boss
  14. Center for Microscopy and Image Analysis, University of Zürich, Zürich, Switzerland.

    • Andres Kaech
  15. Institute of Neurology Clinical Center of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia.

    • Valerija Dobričić,
    • Igor Petrović,
    • Milena Janković,
    • Ivana Novaković &
    • Vladimir S Kostić
  16. Department of Medicine (Neurology), University of Toronto, Toronto, Ontario, Canada.

    • Janis M Miyasaki
  17. Human Genome Study Center, University of São Paulo, São Paulo, Brazil.

    • Mayana Zatz
  18. Klinikum Aschaffenburg, Aschaffenburg, Germany.

    • Jörg Klepper
  19. Department of Pathology and Laboratory Science, Cedars-Sinai Medical Center, Los Angeles, California, USA.

    • Elizabeth Spiteri
  20. Department of Neurology, Clinical University Hospital, Santiago de Compostela, Spain.

    • Jose M Prieto
  21. Department of Neurology, Fundación Jiménez-Díaz, Madrid, Spain.

    • Inmaculada Navas
  22. Institute for Medical Biometry and Statistics, University of Lübeck, Lübeck, Germany.

    • Michael Preuss &
    • Carmen Dering
  23. Translational Neuropharmacology, Clinical Neuroscience, Center for Molecular Medicine, Karolinska Institutet and Neurology Clinic, Karolinska University Hospital, Huddinge, Stockholm, Sweden.

    • Martin Paucar &
    • Per Svenningsson
  24. Department of Reproductive Genetics and Biotechnology, Reproductive Biotechnology Research Center, Avicenna Research Institute, Academic Center for Education, Culture and Research (ACECR), Tehran, Iran.

    • Kioomars Saliminejad
  25. Genetic Research Center, University of Social Welfare and Rehabilitation Science, Tehran, Iran.

    • Hamid R K Khorshid
  26. INSERM Unité Mixte de Recherche Scientifique (UMRS) 975, Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière (CRICM), Paris, France.

    • Isabelle Le Ber
  27. Université Pierre et Marie Curie, Université Paris 6, UMRS 975, Paris, France.

    • Isabelle Le Ber
  28. Centre National de la Recherche Scientifique (CNRS) UMR 7225, Paris, France.

    • Isabelle Le Ber
  29. Assistance Publique–Hôpitaux de Paris (AP-HP), Pitié-Salpêtrière Hospital, Centre de Référence des Démences Rares, Paris, France.

    • Isabelle Le Ber
  30. Department of Neurology, Caen University Hospital, Caen, France.

    • Gilles Defer
  31. Department of Research, Rouvray Psychiatric Hospital, Sotteville-lès-Rouen, France.

    • Dominique Campion
  32. Program in Neurobehavioral Genetics, Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

    • Giovanni Coppola
  33. Neuropsychiatry Department, Federal University of Pernambuco, Recife, Brazil.

    • Joao R M Oliveira

Contributions

J.R.M.O., C.B., C.K. and V.S.K. initiated the project, which was subsequently developed and led jointly by A. Keller, A.W., M.J.S., C.B., C.K. and J.R.M.O. A. Keller, A.W., M.J.S., K.L., K.Z., I. Navas, C.B., C.K. and J.R.M.O. conceived the experiments. A. Keller, E.J.R., M.H., R.R., I.A., M.A.M., E.R., M.C.W., A.B. and A. Kaech performed the mouse experiments, which were financially supported by C.B. and A.A. A.W., M.J.S., M.G.-M., A.D., R.L.S., R.R.L., A.O.-U., G.N., J.E.G.d.C., K.L., V.D., A.C., I.P., J.M.M., M.Z., K.Z., J.K., E.S., J.M.P., I. Navas, M. Preuss, C.D., M.J., M. Paucar, P.S., K.S., H.R.K.K., I. Novaković, I.L.B., G.D., D.H., V.S.K., D.C., D.H.G., G.C., C.K. and J.R.M.O. recruited and examined patients, collected and analyzed human DNA and/or interpreted genetic data. C.B., A. Keller, A.W., M.J.S., C.K. and J.R.M.O. wrote the manuscript with critical input from A.D., K.L. and K.Z.

Competing financial interests

The authors declare no competing financial interests.

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