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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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Related in: MedlinePlus

In situ ceramide localization and sphingolipid profile of cultured keratinocytes.(A) Diagram depicting the de novo synthesis pathway of (glucosyl)ceramides. Loss-of-function of ceramide synthase 1–6 results in an impaired N-acylation of dihydrosphingosine as indicated by a cross. (B) Confocal microscopy images of healthy control and patient H skin biopsies immunostained with an antibody targeting ceramides (red) with DAPI (blue) as nuclear counterstaining. Ceramides localize to the stratum granulosum and the stratum corneum in healthy control skin. Note the loss of ceramide staining in patient's skin. We observed immunostaining of ceramides in the uppermost layer of the mutant stratum corneum, which results from unspecific binding of the secondary antibody. 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) Upper panel: TLC of lipid extracts from healthy control and patient H keratinocytes 14 days after induction of differentiation. Lipids corresponding to 400 µg of cellular protein were extracted from cultures, separated twice by TLC using the solvent system chloroform/methanol/glacial acetic acid (190/9/1 v/v/v), and quantified after carbonization. An epidermal lipid extract of a healthy control individual was used as a reference. Lower panel: Data are presented as mean values +S.D. of triplicate samples and are representative for three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test (* p<0.05, ** p<0.01, *** p<0.001). Ceramide species are classified according to the sphingoid base (S, sphingosine or P, phytosphingosine). Abbreviations: AcylCer, acylceramides; DAG, diacylglycerols; GlcAcylCer, glucosylacylceramides; GlcCer, glucosylceramides; MAG, monoacylglycerols; M/LC-Cer, middle and long-chain ceramides; NEFA, non-esterified fatty acids; TAG, triacylglycerols; VLC-Cer, very long-chain ceramides.
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pgen-1003536-g004: In situ ceramide localization and sphingolipid profile of cultured keratinocytes.(A) Diagram depicting the de novo synthesis pathway of (glucosyl)ceramides. Loss-of-function of ceramide synthase 1–6 results in an impaired N-acylation of dihydrosphingosine as indicated by a cross. (B) Confocal microscopy images of healthy control and patient H skin biopsies immunostained with an antibody targeting ceramides (red) with DAPI (blue) as nuclear counterstaining. Ceramides localize to the stratum granulosum and the stratum corneum in healthy control skin. Note the loss of ceramide staining in patient's skin. We observed immunostaining of ceramides in the uppermost layer of the mutant stratum corneum, which results from unspecific binding of the secondary antibody. 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) Upper panel: TLC of lipid extracts from healthy control and patient H keratinocytes 14 days after induction of differentiation. Lipids corresponding to 400 µg of cellular protein were extracted from cultures, separated twice by TLC using the solvent system chloroform/methanol/glacial acetic acid (190/9/1 v/v/v), and quantified after carbonization. An epidermal lipid extract of a healthy control individual was used as a reference. Lower panel: Data are presented as mean values +S.D. of triplicate samples and are representative for three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test (* p<0.05, ** p<0.01, *** p<0.001). Ceramide species are classified according to the sphingoid base (S, sphingosine or P, phytosphingosine). Abbreviations: AcylCer, acylceramides; DAG, diacylglycerols; GlcAcylCer, glucosylacylceramides; GlcCer, glucosylceramides; MAG, monoacylglycerols; M/LC-Cer, middle and long-chain ceramides; NEFA, non-esterified fatty acids; TAG, triacylglycerols; VLC-Cer, very long-chain ceramides.

Mentions: Since CERS3 generates epidermis-specific ceramides by N-acylating dihydrosphingosine with acyl-CoAs ranging from long to very long aliphatic chains (C18–C28) [11], [12] (Figure 4A), we examined whether mutated CERS3 affects the localization and concentration of ceramides in human epidermis using a commercially available antibody targeting ceramides [13]. Immunofluorescence analysis on frozen sections of a control skin biopsy demonstrated the presence of ceramides in the stratum granulosum and stratum corneum, consistent with the localization of CERS3. In the skin biopsy of the patient carrying the CERS3 splice donor site mutation, however, we detected a massive reduction of ceramides in these layers (Figure 4B). To study the disturbed sphingolipid profile of the patient keratinocytes in detail, we performed TLC analysis of lipid extracts from differentiated control and mutant cells (Figure 4C and Figure S4). Compatible with the results of immunofluorescence staining, lipid extracts of patient keratinocytes compared to control samples exhibited a marked decrease of very long-chain (VLC) ceramides with sphingosine (−48.2%) and phytosphingosine (−47.9%) as sphingoid base as well as significantly decreased levels of acylceramides (−49.9%), glucosylacylceramides (−95.9%), and glucosylceramides (−60.2%). In contrast, levels of ceramides with middle to long-chain acyl moieties (C16–20) were slightly but significantly increased (1.4-fold for sphingosine and 1.2-fold for phytosphingosine as sphingoid base) in patient keratinocytes compared to control samples.


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)

In situ ceramide localization and sphingolipid profile of cultured keratinocytes.(A) Diagram depicting the de novo synthesis pathway of (glucosyl)ceramides. Loss-of-function of ceramide synthase 1–6 results in an impaired N-acylation of dihydrosphingosine as indicated by a cross. (B) Confocal microscopy images of healthy control and patient H skin biopsies immunostained with an antibody targeting ceramides (red) with DAPI (blue) as nuclear counterstaining. Ceramides localize to the stratum granulosum and the stratum corneum in healthy control skin. Note the loss of ceramide staining in patient's skin. We observed immunostaining of ceramides in the uppermost layer of the mutant stratum corneum, which results from unspecific binding of the secondary antibody. 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) Upper panel: TLC of lipid extracts from healthy control and patient H keratinocytes 14 days after induction of differentiation. Lipids corresponding to 400 µg of cellular protein were extracted from cultures, separated twice by TLC using the solvent system chloroform/methanol/glacial acetic acid (190/9/1 v/v/v), and quantified after carbonization. An epidermal lipid extract of a healthy control individual was used as a reference. Lower panel: Data are presented as mean values +S.D. of triplicate samples and are representative for three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test (* p<0.05, ** p<0.01, *** p<0.001). Ceramide species are classified according to the sphingoid base (S, sphingosine or P, phytosphingosine). Abbreviations: AcylCer, acylceramides; DAG, diacylglycerols; GlcAcylCer, glucosylacylceramides; GlcCer, glucosylceramides; MAG, monoacylglycerols; M/LC-Cer, middle and long-chain ceramides; NEFA, non-esterified fatty acids; TAG, triacylglycerols; VLC-Cer, very long-chain ceramides.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003536-g004: In situ ceramide localization and sphingolipid profile of cultured keratinocytes.(A) Diagram depicting the de novo synthesis pathway of (glucosyl)ceramides. Loss-of-function of ceramide synthase 1–6 results in an impaired N-acylation of dihydrosphingosine as indicated by a cross. (B) Confocal microscopy images of healthy control and patient H skin biopsies immunostained with an antibody targeting ceramides (red) with DAPI (blue) as nuclear counterstaining. Ceramides localize to the stratum granulosum and the stratum corneum in healthy control skin. Note the loss of ceramide staining in patient's skin. We observed immunostaining of ceramides in the uppermost layer of the mutant stratum corneum, which results from unspecific binding of the secondary antibody. 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) Upper panel: TLC of lipid extracts from healthy control and patient H keratinocytes 14 days after induction of differentiation. Lipids corresponding to 400 µg of cellular protein were extracted from cultures, separated twice by TLC using the solvent system chloroform/methanol/glacial acetic acid (190/9/1 v/v/v), and quantified after carbonization. An epidermal lipid extract of a healthy control individual was used as a reference. Lower panel: Data are presented as mean values +S.D. of triplicate samples and are representative for three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t-test (* p<0.05, ** p<0.01, *** p<0.001). Ceramide species are classified according to the sphingoid base (S, sphingosine or P, phytosphingosine). Abbreviations: AcylCer, acylceramides; DAG, diacylglycerols; GlcAcylCer, glucosylacylceramides; GlcCer, glucosylceramides; MAG, monoacylglycerols; M/LC-Cer, middle and long-chain ceramides; NEFA, non-esterified fatty acids; TAG, triacylglycerols; VLC-Cer, very long-chain ceramides.
Mentions: Since CERS3 generates epidermis-specific ceramides by N-acylating dihydrosphingosine with acyl-CoAs ranging from long to very long aliphatic chains (C18–C28) [11], [12] (Figure 4A), we examined whether mutated CERS3 affects the localization and concentration of ceramides in human epidermis using a commercially available antibody targeting ceramides [13]. Immunofluorescence analysis on frozen sections of a control skin biopsy demonstrated the presence of ceramides in the stratum granulosum and stratum corneum, consistent with the localization of CERS3. In the skin biopsy of the patient carrying the CERS3 splice donor site mutation, however, we detected a massive reduction of ceramides in these layers (Figure 4B). To study the disturbed sphingolipid profile of the patient keratinocytes in detail, we performed TLC analysis of lipid extracts from differentiated control and mutant cells (Figure 4C and Figure S4). Compatible with the results of immunofluorescence staining, lipid extracts of patient keratinocytes compared to control samples exhibited a marked decrease of very long-chain (VLC) ceramides with sphingosine (−48.2%) and phytosphingosine (−47.9%) as sphingoid base as well as significantly decreased levels of acylceramides (−49.9%), glucosylacylceramides (−95.9%), and glucosylceramides (−60.2%). In contrast, levels of ceramides with middle to long-chain acyl moieties (C16–20) were slightly but significantly increased (1.4-fold for sphingosine and 1.2-fold for phytosphingosine as sphingoid base) in patient keratinocytes compared to control samples.

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