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

Journal name:
Nature Genetics
Year published:
Published online


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.

At a glance


  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.

Accession codes

Referenced accessions



  1. Oji, V. et al. Revised nomenclature and classification of inherited ichthyoses: results of the First Ichthyosis Consensus Conference in Soreze 2009. J. Am. Acad. Dermatol. 63, 607641 (2010).
  2. Fischer, J. Autosomal recessive congenital ichthyosis. J. Invest. Dermatol. 129, 13191321 (2009).
  3. Israeli, S. et al. A mutation in LIPN, encoding epidermal lipase N, causes a late-onset form of autosomal-recessive congenital ichthyosis. Am. J. Hum. Genet. 88, 482487 (2011).
  4. Lefèvre, C. et al. Mutations in CGI-58, the gene encoding a new protein of the esterase/lipase/thioesterase subfamily, in Chanarin-Dorfman syndrome. Am. J. Hum. Genet. 69, 10021012 (2001).
  5. Demerjian, M., Crumrine, D.A., Milstone, L.M., Williams, M.L. & Elias, P.M. Barrier dysfunction and pathogenesis of neutral lipid storage disease with ichthyosis (Chanarin-Dorfman syndrome). J. Invest. Dermatol. 126, 20322038 (2006).
  6. Yamaguchi, T. & Osumi, T. Chanarin-Dorfman syndrome: deficiency in CGI-58, a lipid droplet-bound coactivator of lipase. Biochim. Biophys. Acta 1791, 519523 (2009).
  7. Zimmermann, R., Lass, A., Haemmerle, G. & Zechner, R. Fate of fat: the role of adipose triglyceride lipase in lipolysis. Biochim. Biophys. Acta 1791, 494500 (2009).
  8. Schweiger, M. et al. The C-terminal region of human adipose triglyceride lipase affects enzyme activity and lipid droplet binding. J. Biol. Chem. 283, 1721117220 (2008).
  9. Fischer, J. et al. The gene encoding adipose triglyceride lipase (PNPLA2) is mutated in neutral lipid storage disease with myopathy. Nat. Genet. 39, 2830 (2007).
  10. Ohkuma, A. et al. Distal lipid storage myopathy due to PNPLA2 mutation. Neuromuscul. Disord. 18, 671674 (2008).
  11. Akiyama, M. et al. Novel duplication mutation in the patatin domain of adipose triglyceride lipase (PNPLA2) in neutral lipid storage disease with severe myopathy. Muscle Nerve 36, 856859 (2007).
  12. Lake, A.C. et al. Expression, regulation, and triglyceride hydrolase activity of Adiponutrin family members. J. Lipid Res. 46, 24772487 (2005).
  13. Wilson, P.A., Gardner, S.D., Lambie, N.M., Commans, S.A. & Crowther, D.J. Characterization of the human patatin-like phospholipase family. J. Lipid Res. 47, 19401949 (2006).
  14. Kienesberger, P.C., Oberer, M., Lass, A. & Zechner, R. Mammalian patatin domain containing proteins: a family with diverse lipolytic activities involved in multiple biological functions. J. Lipid Res. 50 (suppl.), S63S68 (2009).
  15. Baulande, S. & Langlois, C. Proteins sharing PNPLA domain, a new family of enzymes regulating lipid metabolism. Med. Sci. (Paris) 26, 177184 (2010).
  16. Romeo, S. et al. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat. Genet. 40, 14611465 (2008).
  17. Tian, C., Stokowski, R.P., Kershenobich, D., Ballinger, D.G. & Hinds, D.A. Variant in PNPLA3 is associated with alcoholic liver disease. Nat. Genet. 42, 2123 (2010).
  18. Rainier, S. et al. Neuropathy target esterase gene mutations cause motor neuron disease. Am. J. Hum. Genet. 82, 780785 (2008).
  19. Mubaidin, A. et al. Karak syndrome: a novel degenerative disorder of the basal ganglia and cerebellum. J. Med. Genet. 40, 543546 (2003).
  20. Tan, E.K., Ho, P., Tan, L., Prakash, K.M. & Zhao, Y. PLA2G6 mutations and Parkinson's disease. Ann. Neurol. 67, 148 (2010).
  21. Gregory, A. et al. Neurodegeneration associated with genetic defects in phospholipase A(2). Neurology 71, 14021409 (2008).
  22. Sutter, N.B. & Ostrander, E.A. Dog star rising: the canine genetic system. Nat. Rev. Genet. 5, 900910 (2004).
  23. Karlsson, E.K. et al. Efficient mapping of mendelian traits in dogs through genome-wide association. Nat. Genet. 39, 13211328 (2007).
  24. Galibert, F. & Andre, C. The dog: a powerful model for studying genotype-phenotype relationships. Comp. Biochem. Physiol. Part D Genomics Proteomics 3, 6777 (2008).
  25. Cadieu, E. et al. Coat variation in the domestic dog is governed by variants in three genes. Science 326, 150153 (2009).
  26. Parker, H.G. et al. An expressed fgf4 retrogene is associated with breed-defining chondrodysplasia in domestic dogs. Science 325, 995998 (2009).
  27. Merveille, A.C. et al. CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs. Nat. Genet. 43, 7278 (2011).
  28. Credille, K.M., Barnhart, K.F., Minor, J.S. & Dunstan, R.W. Mild recessive epidermolytic hyperkeratosis associated with a novel keratin 10 donor splice-site mutation in a family of Norfolk terrier dogs. Br. J. Dermatol. 153, 5158 (2005).
  29. Credille, K.M. et al. Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs. Br. J. Dermatol. 161, 265272 (2009).
  30. Akiyama, M. & Shimizu, H. An update on molecular aspects of the non-syndromic ichthyoses. Exp. Dermatol. 17, 373382 (2008).
  31. Huber, M. et al. Mutations of keratinocyte transglutaminase in lamellar ichthyosis. Science 267, 525528 (1995).
  32. Parmentier, L. et al. Autosomal recessive lamellar ichthyosis: identification of a new mutation in transglutaminase 1 and evidence for genetic heterogeneity. Hum. Mol. Genet. 4, 13911395 (1995).
  33. Russell, L.J. et al. Mutations in the gene for transglutaminase 1 in autosomal recessive lamellar ichthyosis. Nat. Genet. 9, 279283 (1995).
  34. Mauldin, E.A., Credille, K.M., Dunstan, R.W. & Casal, M.L. Clinical, histopathological and ultrastructural analysis of golden retriever ichthyosis. Vet. Dermatol. 18, 187 (2007).
  35. Guaguere, E., Bensignor, E., Muller, A., Degorce-Rubiales, F. & Andre, C. Epidemiological, clinical, histopathological and ultrastructural aspects of ichthyosis in golden retrievers: a report of 50 cases. Vet. Dermatol. 18, 382383 (2007).
  36. Cadiergues, M.C. et al. Cornification defect in the golden retriever: clinical, histopathological, ultrastructural and genetic characterisation. Vet. Dermatol. 19, 120129 (2008).
  37. Mauldin, E.A., Credille, K.M., Dunstan, R.W. & Casal, M.L. The clinical and morphologic features of nonepidermolytic ichthyosis in the golden retriever. Vet. Pathol. 45, 174180 (2008).
  38. Guaguere, E. et al. Clinical, histopathological and genetic data of ichthyosis in the golden retriever: a prospective study. J. Small Anim. Pract. 50, 227235 (2009).
  39. Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559575 (2007).
  40. Adzhubei, I.A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248249 (2010).
  41. Akiyama, M. et al. CGI-58 is an alpha/beta-hydrolase within lipid transporting lamellar granules of differentiated keratinocytes. Am. J. Pathol. 173, 13491360 (2008).
  42. Eckert, R.L. et al. Regulation of involucrin gene expression. J. Invest. Dermatol. 123, 1322 (2004).
  43. Hitomi, K. Transglutaminases in skin epidermis. Eur. J. Dermatol. 15, 313319 (2005).
  44. Toulza, E. et al. Large-scale identification of human genes implicated in epidermal barrier function. Genome Biol. 8, R107 (2007).
  45. Elias, P.M., Williams, M.L., Holleran, W.M., Jiang, Y.J. & Schmuth, M. Pathogenesis of permeability barrier abnormalities in the ichthyoses: inherited disorders of lipid metabolism. J. Lipid Res. 49, 697714 (2008).
  46. Laiho, E. et al. Clinical and morphological correlations for transglutaminase 1 gene mutations in autosomal recessive congenital ichthyosis. Eur. J. Hum. Genet. 7, 625632 (1999).
  47. Klar, J. et al. Mutations in the fatty acid transport protein 4 gene cause the ichthyosis prematurity syndrome. Am. J. Hum. Genet. 85, 248253 (2009).
  48. Ziblat, R., Leiserowitz, L. & Addadi, L. Crystalline domain structure and cholesterol crystal nucleation in single hydrated DPPC:cholesterol:POPC bilayers. J. Am. Chem. Soc. 132, 99209927 (2010).
  49. Johansson, L.E. et al. Genetic variance in the adiponutrin gene family and childhood obesity. PLoS ONE 4, e5327 (2009).
  50. Gao, J.G., Shih, A., Gruber, R., Schmuth, M. & Simon, M. GS2 as a retinol transacylase and as a catalytic dyad independent regulator of retinylester accretion. Mol. Genet. Metab. 96, 253260 (2009).
  51. Breiden, B., Gallala, H., Doering, T. & Sandhoff, K. Optimization of submerged keratinocyte cultures for the synthesis of barrier ceramides. Eur. J. Cell Biol. 86, 657673 (2007).
  52. Slot, J.W. & Geuze, H.J. Cryosectioning and immunolabeling. Nat. Protoc. 2, 24802491 (2007).
  53. Lass, A. et al. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome. Cell Metab. 3, 309319 (2006).
  54. Zimmermann, R. et al. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase. Science 306, 13831386 (2004).

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Author information

  1. These authors jointly directed this work.

    • Catherine André &
    • Judith Fischer


  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


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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