PNPLA1 mutations cause autosomal recessive congenital ichthyosis in golden retriever dogs and humans

Journal name:
Nature Genetics
Volume:
44,
Pages:
140–147
Year published:
DOI:
doi:10.1038/ng.1056
Received
Accepted
Published online

Abstract

Ichthyoses comprise a heterogeneous group of genodermatoses characterized by abnormal desquamation over the whole body, for which the genetic causes of several human forms remain unknown. We used a spontaneous dog model in the golden retriever breed, which is affected by a lamellar ichthyosis resembling human autosomal recessive congenital ichthyoses (ARCI), to carry out a genome-wide association study. We identified a homozygous insertion-deletion (indel) mutation in PNPLA1 that leads to a premature stop codon in all affected golden retriever dogs. We subsequently found one missense and one nonsense mutation in the catalytic domain of human PNPLA1 in six individuals with ARCI from two families. Further experiments highlighted the importance of PNPLA1 in the formation of the epidermal lipid barrier. This study identifies a new gene involved in human ichthyoses and provides insights into the localization and function of this yet uncharacterized member of the PNPLA protein family.

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Figures

  1. Identification of the PNPLA1 mutation in affected golden retriever dogs.
    Figure 1: Identification of the PNPLA1 mutation in affected golden retriever dogs.

    (a) In these dogs, generalized scaling, with white or blackish scales, and large ichthyosiform adherent scales are suggestive of ichthyosis. (b) The structure of the dog PNPLA1 gene. Sequencing PNPLA1 in affected dogs revealed an indel in exon 8 indicated by a star. (c) Predicted structure of wild-type and mutant PNPLA1 proteins, including the patatin domain (beginning at Ile16) and hydrophobic domain (ending at Ser409). The mutation induces a frameshift and a premature stop codon. International patent for the detection of the mutation in dogs: PCT/EP2010/067569.

  2. Identification of PNPLA1 mutations in humans with autosomal recessive congenital ichthyosis.
    Figure 2: Identification of PNPLA1 mutations in humans with autosomal recessive congenital ichthyosis.

    (a) Clinical example of ARCI. Two affected sisters (35 and 37 years of age) from the consanguineous Algerian family, born as collodion babies, present similar dermatological characteristics. While they are treated with emollients, the clinical features are the following: nonbullous and nonsyndromic congenital ichthyosiform erythroderma with diffuse mild erythema, mild hyperkeratosis with thin, white and adhesive scales, diffuse even in the flexures, with a reticulated aspect on the back and thighs. Hyperkeratosis is more severe with larger and octagonal scales on the legs. There is a mild palmo-plantar keratoderma, a pseudo-syndactyly of the second and third toes. The older sister has been treated since age 35 with acitretin (0.25 mg/kg/d) and keratolytic topics; her erythroderma is more pronounced but the scaling has totally disappeared across the integument (also see Supplementary Fig. 3). (b) The structure of the human PNPLA1 gene and sites of the mutations (exon 1 and 2) indicated by the stars. (c) Predicted structure of wild-type and mutant PNPLA1 proteins, including the patatin domain (beginning at Ile16) and hydrophobic domain (between Leu336 and Ser418). In family 1, the nonsense mutation c.391G>T leads to a premature stop codon at position 131. In family 2, the missense mutation c.176C>T leads to a p.Ala59Val substitution. Informed consent was obtained from the individuals pictured here.

  3. Histological analysis of skin biopsies from golden retriever dogs and human subjects.
    Figure 3: Histological analysis of skin biopsies from golden retriever dogs and human subjects.

    (ad) Hematoxylin and eosin staining of skin biopsies from a healthy dog (a), a dog affected with ichthyosis (original magnification 400×) (b), a healthy human (c) or an individual with ARCI (original magnification 200×) (d). (eh) Light microscopy images of semi-thin sections of skin biopsies stained with methylene blue from a healthy dog (e), a dog affected with ichthyosis (f), a healthy human (g) or an individual with ARCI (h). Biopsies of affected individuals are characterized by a pronounced hyperkeratosis with a homogenous thicker compact orthokeratotic cornified layer as well as a thicker granular layer. Black arrows in f indicate vacuolic structures in keratinocytes from the subgranular layer of affected dog biopsies. Black dotted arrows in h indicate small holes within granular layer of human biopsies. Scale bars, 20 μm.

  4. Localization of wild-type PNPLA1 protein in human skin.
    Figure 4: Localization of wild-type PNPLA1 protein in human skin.

    (ac) Confocal microscopy images of double immunostained human skin paraffin section from a healthy individual for the granular layer marker FLG (filaggrin); green) with DAPI as nuclear counterstaining (blue) (a) and PNPLA1 (red), indicating its expression throughout the epidermis (b). (c) The merged image shows colocalization of the two proteins, suggesting coexpression of PNPLA1 in the granular layer together with filaggrin. Scale bars, 20 μm. (df) Immunoelectron microscopy observations of the PNPLA1 protein in cryo-ultrasection from a healthy individual showing labeling in the regions of keratin filament bundles, predominantly in the upper epidermal layer (as shown by circles). K, keratin filament bundles; KH, keratohyalin; LB, lamellar bodies. Scale bars, 250 nm.

  5. Transmission electron micrographs of fixed fresh skin biopsies from golden retriever dogs and humans.
    Figure 5: Transmission electron micrographs of fixed fresh skin biopsies from golden retriever dogs and humans.

    Electron microscopy of skin biopsies from (a) a healthy dog, (b,c) dogs affected with ichthyosis, (d) a healthy human or (e,f) individuals with ARCI. White arrows indicate pigment granules; black arrows indicate cholesterol clefts in the cornified layer in b and e; black dotted arrows indicate irregular accumulations of abnormal membranous and vesicular material around the nuclei of cells from the granular layer in c and f. Scale bars in ac, 2.5 μm. Scale bars in d and f, 1 μm. Scale bar in e, 0.5 μm.

  6. Protein blotting of PNPLA1 in normal and mutant human keratinocytes, before differentiation and at 3 and 7 d after induction of differentiation.
    Figure 6: Protein blotting of PNPLA1 in normal and mutant human keratinocytes, before differentiation and at 3 and 7 d after induction of differentiation.

    (a) PNPLA1 protein was detected at ~58 kDa using antibody against PNPLA1. To confirm equal protein loading, membranes were stained with Coomassie blue. (b) Detection of proteins was performed using antibodies against transglutaminase 1, involucrin and CGI-58; GAPDH was used as a loading control. Transglutaminase 1 was detected at ~90 kDa, involucrin at ~120 kDa, CGI-58 at ~39 kDa and GAPDH at ~37 kDa.

  7. Triglyceride hydrolase activity and lipid profiles of wild-type and PNPLA1-deficient human keratinocytes in cell culture.
    Figure 7: Triglyceride hydrolase activity and lipid profiles of wild-type and PNPLA1-deficient human keratinocytes in cell culture.

    (a) Neutral lipid profiles of wild-type and mutant human keratinocytes 7 d after induction of differentiation in culture. Lipids corresponding to 300 μg of protein were extracted and separated by thin-layer chromatography (TLC) using hexan/diethyl ether/glacial acetic acid (70:29:1) as solvent system. Spots were visualized by carbonization. (b) Triglyceride hydrolase activity of PNPLA1 and PNPLA2 in COS-7 cells transfected to express PNPLA1 and PNPLA2, with or without CGI-58, and measured by the release of radiolabeled fatty acids. β-galactosidase–transfected cells were used as the negative control and were set as the blank. (c) Incorporation of [14C]-linoleic acid into phospholipids of differentiated wild-type and mutant human keratinocytes visualized after TLC by exposure to a phosphorimager screen and quantified with ImageQuant software. Data are presented as mean ± s.d. Statistical significance was determined by unpaired two-tailed Student's t test (*P < 0.05; ***P < 0.001). CE, cholesterol ester; TG, triglycerides; FFA, free fatty acids; Chol., cholesterol; DG, diglycerides; PL, phospholipids; PA, phosphatidic acid; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine.

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Author information

  1. These authors jointly directed this work.

    • Catherine André &
    • Judith Fischer

Affiliations

  1. Centre National de la Recherche Scientifique (CNRS), Institut de Génétique et Développement de Rennes, Rennes, France.

    • Anaïs Grall,
    • Sandrine Planchais,
    • Christophe Hitte,
    • Matthieu Le Gallo,
    • Laëtitia Lagoutte,
    • Sébastien Küry,
    • Francis Galibert &
    • Catherine André
  2. Université Rennes 1, Institut Fédératif de Recherche (IFR) 140, Faculté de Médecine, Rennes, France.

    • Anaïs Grall,
    • Sandrine Planchais,
    • Christophe Hitte,
    • Matthieu Le Gallo,
    • Laëtitia Lagoutte,
    • Sébastien Küry,
    • Francis Galibert &
    • Catherine André
  3. Clinique Vétérinaire Saint Bernard, Lomme, France.

    • Eric Guaguère
  4. Institute of Molecular Biosciences, Karl-Franzens-Universität Graz, Graz, Austria.

    • Susanne Grond,
    • Franz P W Radner,
    • Robert Zimmermann &
    • Rudolf Zechner
  5. Département de Dermatologie, Hôpital St. Louis, Paris, France.

    • Emmanuelle Bourrat
  6. Department of Dermatology, University Clinic Heidelberg, Heidelberg, Germany.

    • Ingrid Hausser
  7. Electron Microscopy Core Facility University Heidelberg, Heidelberg, Germany.

    • Ingrid Hausser
  8. Institut de Génomique, Centre National de Génotypage (CNG), Commissariat à l'Enérgie Atomique et aux Enérgies Alternatives (CEA), Evry, France.

    • Céline Derbois,
    • Mark Lathrop &
    • Judith Fischer
  9. Institute for Human Genetics, University Clinic Freiburg, Freiburg, Germany.

    • Gwang-Jin Kim &
    • Judith Fischer
  10. Faculty for Biology, University of Freiburg, Freiburg, Germany.

    • Gwang-Jin Kim
  11. Laboratoire d'Anatomie Pathologique Vétérinaire du Sud-Ouest, Toulouse, France.

    • Frédérique Degorce-Rubiales
  12. Antagene, Animal Genetics Laboratory, Limonest, France.

    • Anne Thomas
  13. Centre Hospitalier Universitaire (CHU) Nantes, Service de Génétique Médicale, Nantes, France.

    • Sébastien Küry
  14. Clinique Vétérinaire de la Boulais, Cesson-Sévigné, France.

    • Emmanuel Bensignor
  15. Clinique Vétérinaire, Brussels, Belgium.

    • Jacques Fontaine
  16. Unité de Dermatologie, VetAgro Sup Campus Vétérinaire de Lyon, Marcy l'Etoile, France.

    • Didier Pin
  17. Fondation Jean Dausset–Centre d'Etude de Polymorphisme Humain (CEPH), Paris, France.

    • Mark Lathrop
  18. Zentrum für Biosystemanalyse, Universität Freiburg, Freiburg, Germany.

    • Judith Fischer

Contributions

C.A., E.G. and F.G. designed the genetic aspects of the dog experiments. A.G., S.P., C.H., M.L.G., L.L. and S.K. performed the genetic and functional experiments for the dog studies. J. Fischer designed the human genetic analyses and supervised the functional studies on humans. E. Bourrat provided patient material and data. C.D. and G.-J.K. performed the genetic and microscopy experiments for the human studies. I.H. performed light and electron microscopy as well as immunoelectron microscopy investigations. F.D.-R. did H&E staining for histological diagnosis and investigations in dogs. S.G., F.P.W.R., R. Zimmermann and R. Zechner performed functional studies E.G., E. Bensignor, J. Fontaine and D.P., veterinarians specializing in dermatology, collected dog samples and interpreted clinical and biological data. A.T. provided 400 dog DNA samples and performed validation of the mutation in dogs. C.A., A.G., J. Fischer, F.G., C.H., M.L. and I.H.. contributed to the writing of the manuscript.

Competing financial interests

CNRS and Université Rennes 1 (including C.A., E.G. and S.P.) have applied for an international patent (Catherine André et al., PCT/EP2010/067569) covering the use of the canine PNPLA1 mutation for the genetic screening of ichthyosis in dogs. The Antagene laboratory has the international license for providing the ichthyosis DNA test in dogs.

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