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Mutations in CERS3 cause autosomal recessive congenital ichthyosis in humans.

Radner FP, Marrakchi S, Kirchmeier P, Kim GJ, Ribierre F, Kamoun B, Abid L, Leipoldt M, Turki H, Schempp W, Heilig R, Lathrop M, Fischer J - PLoS Genet. (2013)

Bottom Line: However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in CERS3 indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients.Functional analysis of patient skin and in vitro differentiated keratinocytes demonstrated that mutations in CERS3 lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation.Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Human Genetics, University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
Autosomal recessive congenital ichthyosis (ARCI) is a rare genetic disorder of the skin characterized by abnormal desquamation over the whole body. In this study we report four patients from three consanguineous Tunisian families with skin, eye, heart, and skeletal anomalies, who harbor a homozygous contiguous gene deletion syndrome on chromosome 15q26.3. Genome-wide SNP-genotyping revealed a homozygous region in all affected individuals, including the same microdeletion that partially affects two coding genes (ADAMTS17, CERS3) and abolishes a sequence for a long non-coding RNA (FLJ42289). Whereas mutations in ADAMTS17 have recently been identified in autosomal recessive Weill-Marchesani-like syndrome in humans and dogs presenting with ophthalmologic, cardiac, and skeletal abnormalities, no disease associations have been described for CERS3 (ceramide synthase 3) and FLJ42289 so far. However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in CERS3 indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients. Functional analysis of patient skin and in vitro differentiated keratinocytes demonstrated that mutations in CERS3 lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation. Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.

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Histological analysis and CERS3 protein expression in skin biopsies and cultured keratinocytes from healthy controls and patient H.(A) Hematoxylin and eosin staining (H&E) shows acanthosis with a thickening of the granular layer and psoriasiform epidermal hyperplasia in the patient compared to a healthy control. The inset (same scale) shows the detached stratum corneum of the patient. Scale bars, 50 µm (B) Immunofluorescence staining using a specific antibody for CERS3 (red) and DAPI (blue) as nuclear counterstaining. CERS3 staining is present at the interface between the stratum granulosum and the stratum corneum in control skin but not detectable in the patient skin. The thin dashed lines indicate the interface between the stratum granulosum and the stratum corneum as well as the upper edge of the stratum corneum. Scale bars, 50 µm (C) Western blot analysis of CERS3 in control and patient keratinocytes before differentiation (0 d) and at day 4, 7, and 14 after induction of differentiation. CERS3 was detected at ∼37 kDa (indicated by an asterisk) using an antibody targeting the C-terminus of the protein. An antibody that recognizes actin was used as a loading control.
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pgen-1003536-g003: Histological analysis and CERS3 protein expression in skin biopsies and cultured keratinocytes from healthy controls and patient H.(A) Hematoxylin and eosin staining (H&E) shows acanthosis with a thickening of the granular layer and psoriasiform epidermal hyperplasia in the patient compared to a healthy control. The inset (same scale) shows the detached stratum corneum of the patient. Scale bars, 50 µm (B) Immunofluorescence staining using a specific antibody for CERS3 (red) and DAPI (blue) as nuclear counterstaining. CERS3 staining is present at the interface between the stratum granulosum and the stratum corneum in control skin but not detectable in the patient skin. The thin dashed lines indicate the interface between the stratum granulosum and the stratum corneum as well as the upper edge of the stratum corneum. Scale bars, 50 µm (C) Western blot analysis of CERS3 in control and patient keratinocytes before differentiation (0 d) and at day 4, 7, and 14 after induction of differentiation. CERS3 was detected at ∼37 kDa (indicated by an asterisk) using an antibody targeting the C-terminus of the protein. An antibody that recognizes actin was used as a loading control.

Mentions: To investigate the role of CERS3 mutations in the development of ichthyosis we performed histological and biochemical studies using a skin sample of patient H carrying the splice site mutation in the CERS3 gene. Since we could not exclude that mutations in ADAMTS17 and/or FLJ42289 interfere with skin physiology, we did not include samples from the patients with the gene deletion syndrome in the following experiments. Histological analysis of the skin biopsy from patient H showed acanthosis with thickening of the stratum granulosum, psoriasiform epidermal hyperplasia (Figure 3A), and normal size of the detached stratum corneum (inset Figure 3A). To examine CERS3 distribution in skin we performed immunofluorescence staining of paraffin as well as frozen sections from human control biopsies. Using an antibody targeting an epitope near the N-terminus of CERS3 (amino acid 59–120) we demonstrated that the protein localizes at the interface between the stratum granulosum and the stratum corneum in the epidermis (Figure 3B and Figure S3) in accordance with data of the Human Protein Atlas (http://www.proteinatlas.org). In contrast, the patient's skin biopsy did not show CERS3 staining suggesting that functional protein is not present in mutant epidermis (Figure 3B). We verified these results by immunoblotting of lysates from in vitro differentiated control and patient keratinocytes using an antibody targeting amino acids 370–383 at the C-terminus of CERS3. Immunoblots revealed expression of the protein in control cells at the basal stage (day 0) and increased levels during progression of differentiation (Figure 3C). Concordant with the results of immunofluorescence staining, we did not detect full-length CERS3 or a truncated version of the protein in mutant keratinocytes under basal conditions as well as at later stages of keratinocyte differentiation.


Mutations in CERS3 cause autosomal recessive congenital ichthyosis in humans.

Radner FP, Marrakchi S, Kirchmeier P, Kim GJ, Ribierre F, Kamoun B, Abid L, Leipoldt M, Turki H, Schempp W, Heilig R, Lathrop M, Fischer J - PLoS Genet. (2013)

Histological analysis and CERS3 protein expression in skin biopsies and cultured keratinocytes from healthy controls and patient H.(A) Hematoxylin and eosin staining (H&E) shows acanthosis with a thickening of the granular layer and psoriasiform epidermal hyperplasia in the patient compared to a healthy control. The inset (same scale) shows the detached stratum corneum of the patient. Scale bars, 50 µm (B) Immunofluorescence staining using a specific antibody for CERS3 (red) and DAPI (blue) as nuclear counterstaining. CERS3 staining is present at the interface between the stratum granulosum and the stratum corneum in control skin but not detectable in the patient skin. The thin dashed lines indicate the interface between the stratum granulosum and the stratum corneum as well as the upper edge of the stratum corneum. Scale bars, 50 µm (C) Western blot analysis of CERS3 in control and patient keratinocytes before differentiation (0 d) and at day 4, 7, and 14 after induction of differentiation. CERS3 was detected at ∼37 kDa (indicated by an asterisk) using an antibody targeting the C-terminus of the protein. An antibody that recognizes actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675029&req=5

pgen-1003536-g003: Histological analysis and CERS3 protein expression in skin biopsies and cultured keratinocytes from healthy controls and patient H.(A) Hematoxylin and eosin staining (H&E) shows acanthosis with a thickening of the granular layer and psoriasiform epidermal hyperplasia in the patient compared to a healthy control. The inset (same scale) shows the detached stratum corneum of the patient. Scale bars, 50 µm (B) Immunofluorescence staining using a specific antibody for CERS3 (red) and DAPI (blue) as nuclear counterstaining. CERS3 staining is present at the interface between the stratum granulosum and the stratum corneum in control skin but not detectable in the patient skin. The thin dashed lines indicate the interface between the stratum granulosum and the stratum corneum as well as the upper edge of the stratum corneum. Scale bars, 50 µm (C) Western blot analysis of CERS3 in control and patient keratinocytes before differentiation (0 d) and at day 4, 7, and 14 after induction of differentiation. CERS3 was detected at ∼37 kDa (indicated by an asterisk) using an antibody targeting the C-terminus of the protein. An antibody that recognizes actin was used as a loading control.
Mentions: To investigate the role of CERS3 mutations in the development of ichthyosis we performed histological and biochemical studies using a skin sample of patient H carrying the splice site mutation in the CERS3 gene. Since we could not exclude that mutations in ADAMTS17 and/or FLJ42289 interfere with skin physiology, we did not include samples from the patients with the gene deletion syndrome in the following experiments. Histological analysis of the skin biopsy from patient H showed acanthosis with thickening of the stratum granulosum, psoriasiform epidermal hyperplasia (Figure 3A), and normal size of the detached stratum corneum (inset Figure 3A). To examine CERS3 distribution in skin we performed immunofluorescence staining of paraffin as well as frozen sections from human control biopsies. Using an antibody targeting an epitope near the N-terminus of CERS3 (amino acid 59–120) we demonstrated that the protein localizes at the interface between the stratum granulosum and the stratum corneum in the epidermis (Figure 3B and Figure S3) in accordance with data of the Human Protein Atlas (http://www.proteinatlas.org). In contrast, the patient's skin biopsy did not show CERS3 staining suggesting that functional protein is not present in mutant epidermis (Figure 3B). We verified these results by immunoblotting of lysates from in vitro differentiated control and patient keratinocytes using an antibody targeting amino acids 370–383 at the C-terminus of CERS3. Immunoblots revealed expression of the protein in control cells at the basal stage (day 0) and increased levels during progression of differentiation (Figure 3C). Concordant with the results of immunofluorescence staining, we did not detect full-length CERS3 or a truncated version of the protein in mutant keratinocytes under basal conditions as well as at later stages of keratinocyte differentiation.

Bottom Line: However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in CERS3 indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients.Functional analysis of patient skin and in vitro differentiated keratinocytes demonstrated that mutations in CERS3 lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation.Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Institute for Human Genetics, University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
Autosomal recessive congenital ichthyosis (ARCI) is a rare genetic disorder of the skin characterized by abnormal desquamation over the whole body. In this study we report four patients from three consanguineous Tunisian families with skin, eye, heart, and skeletal anomalies, who harbor a homozygous contiguous gene deletion syndrome on chromosome 15q26.3. Genome-wide SNP-genotyping revealed a homozygous region in all affected individuals, including the same microdeletion that partially affects two coding genes (ADAMTS17, CERS3) and abolishes a sequence for a long non-coding RNA (FLJ42289). Whereas mutations in ADAMTS17 have recently been identified in autosomal recessive Weill-Marchesani-like syndrome in humans and dogs presenting with ophthalmologic, cardiac, and skeletal abnormalities, no disease associations have been described for CERS3 (ceramide synthase 3) and FLJ42289 so far. However, analysis of additional patients with non-syndromic ARCI revealed a splice site mutation in CERS3 indicating that a defect in ceramide synthesis is causative for the present skin phenotype of our patients. Functional analysis of patient skin and in vitro differentiated keratinocytes demonstrated that mutations in CERS3 lead to a disturbed sphingolipid profile with reduced levels of epidermis-specific very long-chain ceramides that interferes with epidermal differentiation. Taken together, these data present a novel pathway involved in ARCI development and, moreover, provide the first evidence that CERS3 plays an essential role in human sphingolipid metabolism for the maintenance of epidermal lipid homeostasis.

Show MeSH
Related in: MedlinePlus