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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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Deletion and CERS3 mutation scheme.(A) Homozygous regions on chromosome 15q26.3 in patients. The smallest common interval is defined by rs2684811 in patient C and rs11247226 in patient S. The genomic deletion characterized by breakpoint spanning PCR (in blue) encompasses 106,960 bp with the borders 100,856,031 to 100,962,985 on chromosome 15 (UCSC hg19, February 2009) in patient D1, D2, C, and S. The splice site mutation in patient H is marked by an asterisk. The diagram shows the deletion and the limiting SNPs of the homozygous regions in patient H, D1, D2, C, and S (not to scale). (B) The scale illustration of the deleted region shows the missing SNPs and genes. The internal limit of the deletion is between SNP rs1080429 and rs7179355. (C) FISH signal pattern in a healthy individual shows the control signal on 15q21.2 in red (digoxigenin-labeled BAC RP11-562A8) and the signal corresponding to 15q26.3 in green (arrow). In patients D1 and D2 the green FISH signal is missing confirming the microdeletion in 15q26.3. (D) Diagram depicting the structure of the human CERS3 gene and the site of mutation in exon 9 (c.609+1G>T) of patient H indicated by an asterisk. Below is illustrated the predicted structure of human CERS3 protein including transmembrane domains (TMD), homeobox, polyglutamic acid region (G), and the TLC (TRAM, LAG1 and CLN8 homology) domain.
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pgen-1003536-g002: Deletion and CERS3 mutation scheme.(A) Homozygous regions on chromosome 15q26.3 in patients. The smallest common interval is defined by rs2684811 in patient C and rs11247226 in patient S. The genomic deletion characterized by breakpoint spanning PCR (in blue) encompasses 106,960 bp with the borders 100,856,031 to 100,962,985 on chromosome 15 (UCSC hg19, February 2009) in patient D1, D2, C, and S. The splice site mutation in patient H is marked by an asterisk. The diagram shows the deletion and the limiting SNPs of the homozygous regions in patient H, D1, D2, C, and S (not to scale). (B) The scale illustration of the deleted region shows the missing SNPs and genes. The internal limit of the deletion is between SNP rs1080429 and rs7179355. (C) FISH signal pattern in a healthy individual shows the control signal on 15q21.2 in red (digoxigenin-labeled BAC RP11-562A8) and the signal corresponding to 15q26.3 in green (arrow). In patients D1 and D2 the green FISH signal is missing confirming the microdeletion in 15q26.3. (D) Diagram depicting the structure of the human CERS3 gene and the site of mutation in exon 9 (c.609+1G>T) of patient H indicated by an asterisk. Below is illustrated the predicted structure of human CERS3 protein including transmembrane domains (TMD), homeobox, polyglutamic acid region (G), and the TLC (TRAM, LAG1 and CLN8 homology) domain.

Mentions: We performed genome-wide SNP-array based homozygosity mapping in 34 consanguineous families with ARCI including three Tunisian families with a syndromic form of ARCI. The clinical features in the four patients (D1, D2, C, and S) include collodion membrane at birth evolving to generalized ARCI, but also short stature, brachydactyly with joint stiffness, microspherophakia, ectopia lentis, mitral valve defects, and multiple nevi (Table 1, Figure 1, and Text S1). These patients all shared a homozygous region on chromosome 15q26.3 with an identical haplotype in the smallest common interval of 1.67 Mb (Figure 2A). Within this region, a homozygous deletion of about 100 Kb was observed between the SNP markers rs1080492 and rs7179355 that encompasses the first three exons of ADAMTS17, the complete sequence of the non-coding RNA FLJ42289, and exon 13 of CERS3 including the 3′UTR (Figure 2B). We confirmed the homozygous deletion in these patients using FISH (Figure 2C) and array CGH (comparative genomic hybridization) analysis (data not shown). A breakpoint spanning PCR followed by sequencing defined the genomic deletion encompassing 106,960 bp (Figure S1). Moreover, sequencing of all exons and exon/intron boundaries of the CERS3 gene showed the absence of PCR amplification of exon 13 and therefore confirmed the genomic deletion in the patients (D1, D2, C, and S). ADAMTS17 was not sequenced. One additional Tunisian individual (H) with isolated ARCI also showed a homozygous region on 15q26.3, but did not carry the genomic deletion described above (Figure 2A). However, sequencing of the CERS3 gene in this patient revealed a homozygous transversion of guanine to thymine affecting the exon 9 splice donor site (c.609+1G>T) (Figure 2D). To analyze the functional effect of this mutation event we performed reverse transcription of mRNA from patient H isolated from in vitro differentiated keratinocytes followed by PCR amplification of the corresponding CERS3 region using specific primers. Separation by agarose gel electrophoresis as well as sequencing of the PCR fragment demonstrated a reduced length of the PCR product due to skipping of exon 9 resulting in an in-frame deletion of 93 bp in the CERS3 coding transcript (Figure S2). MutationTaster software calculated a result score of 0.999 for a probable disease causing mutation event [10]. In addition, this sequence variation was not found in 96 unaffected population-matched control individuals.


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)

Deletion and CERS3 mutation scheme.(A) Homozygous regions on chromosome 15q26.3 in patients. The smallest common interval is defined by rs2684811 in patient C and rs11247226 in patient S. The genomic deletion characterized by breakpoint spanning PCR (in blue) encompasses 106,960 bp with the borders 100,856,031 to 100,962,985 on chromosome 15 (UCSC hg19, February 2009) in patient D1, D2, C, and S. The splice site mutation in patient H is marked by an asterisk. The diagram shows the deletion and the limiting SNPs of the homozygous regions in patient H, D1, D2, C, and S (not to scale). (B) The scale illustration of the deleted region shows the missing SNPs and genes. The internal limit of the deletion is between SNP rs1080429 and rs7179355. (C) FISH signal pattern in a healthy individual shows the control signal on 15q21.2 in red (digoxigenin-labeled BAC RP11-562A8) and the signal corresponding to 15q26.3 in green (arrow). In patients D1 and D2 the green FISH signal is missing confirming the microdeletion in 15q26.3. (D) Diagram depicting the structure of the human CERS3 gene and the site of mutation in exon 9 (c.609+1G>T) of patient H indicated by an asterisk. Below is illustrated the predicted structure of human CERS3 protein including transmembrane domains (TMD), homeobox, polyglutamic acid region (G), and the TLC (TRAM, LAG1 and CLN8 homology) domain.
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Related In: Results  -  Collection

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pgen-1003536-g002: Deletion and CERS3 mutation scheme.(A) Homozygous regions on chromosome 15q26.3 in patients. The smallest common interval is defined by rs2684811 in patient C and rs11247226 in patient S. The genomic deletion characterized by breakpoint spanning PCR (in blue) encompasses 106,960 bp with the borders 100,856,031 to 100,962,985 on chromosome 15 (UCSC hg19, February 2009) in patient D1, D2, C, and S. The splice site mutation in patient H is marked by an asterisk. The diagram shows the deletion and the limiting SNPs of the homozygous regions in patient H, D1, D2, C, and S (not to scale). (B) The scale illustration of the deleted region shows the missing SNPs and genes. The internal limit of the deletion is between SNP rs1080429 and rs7179355. (C) FISH signal pattern in a healthy individual shows the control signal on 15q21.2 in red (digoxigenin-labeled BAC RP11-562A8) and the signal corresponding to 15q26.3 in green (arrow). In patients D1 and D2 the green FISH signal is missing confirming the microdeletion in 15q26.3. (D) Diagram depicting the structure of the human CERS3 gene and the site of mutation in exon 9 (c.609+1G>T) of patient H indicated by an asterisk. Below is illustrated the predicted structure of human CERS3 protein including transmembrane domains (TMD), homeobox, polyglutamic acid region (G), and the TLC (TRAM, LAG1 and CLN8 homology) domain.
Mentions: We performed genome-wide SNP-array based homozygosity mapping in 34 consanguineous families with ARCI including three Tunisian families with a syndromic form of ARCI. The clinical features in the four patients (D1, D2, C, and S) include collodion membrane at birth evolving to generalized ARCI, but also short stature, brachydactyly with joint stiffness, microspherophakia, ectopia lentis, mitral valve defects, and multiple nevi (Table 1, Figure 1, and Text S1). These patients all shared a homozygous region on chromosome 15q26.3 with an identical haplotype in the smallest common interval of 1.67 Mb (Figure 2A). Within this region, a homozygous deletion of about 100 Kb was observed between the SNP markers rs1080492 and rs7179355 that encompasses the first three exons of ADAMTS17, the complete sequence of the non-coding RNA FLJ42289, and exon 13 of CERS3 including the 3′UTR (Figure 2B). We confirmed the homozygous deletion in these patients using FISH (Figure 2C) and array CGH (comparative genomic hybridization) analysis (data not shown). A breakpoint spanning PCR followed by sequencing defined the genomic deletion encompassing 106,960 bp (Figure S1). Moreover, sequencing of all exons and exon/intron boundaries of the CERS3 gene showed the absence of PCR amplification of exon 13 and therefore confirmed the genomic deletion in the patients (D1, D2, C, and S). ADAMTS17 was not sequenced. One additional Tunisian individual (H) with isolated ARCI also showed a homozygous region on 15q26.3, but did not carry the genomic deletion described above (Figure 2A). However, sequencing of the CERS3 gene in this patient revealed a homozygous transversion of guanine to thymine affecting the exon 9 splice donor site (c.609+1G>T) (Figure 2D). To analyze the functional effect of this mutation event we performed reverse transcription of mRNA from patient H isolated from in vitro differentiated keratinocytes followed by PCR amplification of the corresponding CERS3 region using specific primers. Separation by agarose gel electrophoresis as well as sequencing of the PCR fragment demonstrated a reduced length of the PCR product due to skipping of exon 9 resulting in an in-frame deletion of 93 bp in the CERS3 coding transcript (Figure S2). MutationTaster software calculated a result score of 0.999 for a probable disease causing mutation event [10]. In addition, this sequence variation was not found in 96 unaffected population-matched control individuals.

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