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Mutations in human C2CD3 cause skeletal dysplasia and provide new insights into phenotypic and cellular consequences of altered C2CD3 function.

Cortés CR, McInerney-Leo AM, Vogel I, Rondón Galeano MC, Leo PJ, Harris JE, Anderson LK, Keith PA, Brown MA, Ramsing M, Duncan EL, Zankl A, Wicking C - Sci Rep (2016)

Bottom Line: Ciliopathies are clinically grouped in a large number of overlapping disorders, including the orofaciodigital syndromes (OFDS), the short rib polydactyly syndromes and Jeune asphyxiating thoracic dystrophy.Analysis of fibroblast cultures derived from one of these fetuses revealed a reduced ability to form cilia, consistent with previous studies in C2cd3-mutant mouse and chicken cells.More detailed analyses support a role for C2CD3 in basal body maturation; but in contrast to previous mouse studies the normal recruitment of the distal appendage protein CEP164 suggests that this protein is not sufficient for efficient basal body maturation and subsequent axonemal extension in a C2CD3-defective background.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT
Ciliopathies are a group of genetic disorders caused by defective assembly or dysfunction of the primary cilium, a microtubule-based cellular organelle that plays a key role in developmental signalling. Ciliopathies are clinically grouped in a large number of overlapping disorders, including the orofaciodigital syndromes (OFDS), the short rib polydactyly syndromes and Jeune asphyxiating thoracic dystrophy. Recently, mutations in the gene encoding the centriolar protein C2CD3 have been described in two families with a new sub-type of OFDS (OFD14), with microcephaly and cerebral malformations. Here we describe a third family with novel compound heterozygous C2CD3 mutations in two fetuses with a different clinical presentation, dominated by skeletal dysplasia with no microcephaly. Analysis of fibroblast cultures derived from one of these fetuses revealed a reduced ability to form cilia, consistent with previous studies in C2cd3-mutant mouse and chicken cells. More detailed analyses support a role for C2CD3 in basal body maturation; but in contrast to previous mouse studies the normal recruitment of the distal appendage protein CEP164 suggests that this protein is not sufficient for efficient basal body maturation and subsequent axonemal extension in a C2CD3-defective background.

No MeSH data available.


Related in: MedlinePlus

Centriolar appendage and basal body maturation in C2CD3-mutant fibroblasts.(a,b) Images of CP110 staining (green) in serum starved control and G3P1 fibroblasts. Note that the image in panel (b) with CP110 at both centrioles is representative of approximately 77% of cells in G3P1cultures (23% of cells have CP110 removed from the mother centriole, i.e. show staining at just one centriole). This quantification is shown graphically in (c) where random cells across both mutant and control cultures with 2 distinct centriolar spots were scored for removal of CP110 from the mother centriole. CP110 is removed in fewer mutant cells relative to control cells. *p < 0.05. (d–g) Representative images of staining for the distal appendage marker CEP164 (green) in non-ciliated (d,e) and ciliated cells (f,g). (h) Quantification of cells with CEP164-positive mother centriole or basal body. (i–l) Representative images of staining for the sub-distal appendage marker ODF2 (green) in non-ciliated (i–j) and ciliated cells (k,l). (m) Quantification of cells with ODF2-positive mother centriole or basal body. Scale bar = 5 μm. In all cases the centrosome is marked with gamma-tubulin and the axoneme with Arl13b, both in red. n.s. not significant, error bars show SEM.
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f4: Centriolar appendage and basal body maturation in C2CD3-mutant fibroblasts.(a,b) Images of CP110 staining (green) in serum starved control and G3P1 fibroblasts. Note that the image in panel (b) with CP110 at both centrioles is representative of approximately 77% of cells in G3P1cultures (23% of cells have CP110 removed from the mother centriole, i.e. show staining at just one centriole). This quantification is shown graphically in (c) where random cells across both mutant and control cultures with 2 distinct centriolar spots were scored for removal of CP110 from the mother centriole. CP110 is removed in fewer mutant cells relative to control cells. *p < 0.05. (d–g) Representative images of staining for the distal appendage marker CEP164 (green) in non-ciliated (d,e) and ciliated cells (f,g). (h) Quantification of cells with CEP164-positive mother centriole or basal body. (i–l) Representative images of staining for the sub-distal appendage marker ODF2 (green) in non-ciliated (i–j) and ciliated cells (k,l). (m) Quantification of cells with ODF2-positive mother centriole or basal body. Scale bar = 5 μm. In all cases the centrosome is marked with gamma-tubulin and the axoneme with Arl13b, both in red. n.s. not significant, error bars show SEM.

Mentions: Defective axonemal extension is a common consequence of altered IFT, and accordingly C2cd3-mutant mouse cells fail to recruit a number of IFT proteins to the centrosome23. The core anterograde IFT protein IFT88 is essential for ciliogenesis and is normally localised to centrioles prior to cilia assembly, and to the transition zone and along the axoneme in ciliated cells31. Consistent with the studies in mouse cells, we observed a defect in the localisation of IFT88 at the centrioles of non-ciliated serum-starved G3P1 mutant cells (Fig. 3d–j), while all ciliated cells recruited IFT88 normally. In addition to IFT, axonemal extension is also dependent on uncapping of the distal end of the mother centriole by removal of CP1107. CP110 removal is defective in C2cd3-mutant mouse cells23, and in agreement with this we found that CP110 was not efficiently removed from the mother centriole in serum-starved G3P1 cells relative to control fibroblast cultures (23% cells in mutant cultures versus 58% in control cultures show CP110 removal; i.e CP110 at only one centriole; Fig. 4a–c). This suggests that C2CD3-mutant G3P1 cells do not efficiently uncap the mother centriole, one of the earliest steps in ciliogenesis, even when ciliogenesis is induced by serum starvation.


Mutations in human C2CD3 cause skeletal dysplasia and provide new insights into phenotypic and cellular consequences of altered C2CD3 function.

Cortés CR, McInerney-Leo AM, Vogel I, Rondón Galeano MC, Leo PJ, Harris JE, Anderson LK, Keith PA, Brown MA, Ramsing M, Duncan EL, Zankl A, Wicking C - Sci Rep (2016)

Centriolar appendage and basal body maturation in C2CD3-mutant fibroblasts.(a,b) Images of CP110 staining (green) in serum starved control and G3P1 fibroblasts. Note that the image in panel (b) with CP110 at both centrioles is representative of approximately 77% of cells in G3P1cultures (23% of cells have CP110 removed from the mother centriole, i.e. show staining at just one centriole). This quantification is shown graphically in (c) where random cells across both mutant and control cultures with 2 distinct centriolar spots were scored for removal of CP110 from the mother centriole. CP110 is removed in fewer mutant cells relative to control cells. *p < 0.05. (d–g) Representative images of staining for the distal appendage marker CEP164 (green) in non-ciliated (d,e) and ciliated cells (f,g). (h) Quantification of cells with CEP164-positive mother centriole or basal body. (i–l) Representative images of staining for the sub-distal appendage marker ODF2 (green) in non-ciliated (i–j) and ciliated cells (k,l). (m) Quantification of cells with ODF2-positive mother centriole or basal body. Scale bar = 5 μm. In all cases the centrosome is marked with gamma-tubulin and the axoneme with Arl13b, both in red. n.s. not significant, error bars show SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Centriolar appendage and basal body maturation in C2CD3-mutant fibroblasts.(a,b) Images of CP110 staining (green) in serum starved control and G3P1 fibroblasts. Note that the image in panel (b) with CP110 at both centrioles is representative of approximately 77% of cells in G3P1cultures (23% of cells have CP110 removed from the mother centriole, i.e. show staining at just one centriole). This quantification is shown graphically in (c) where random cells across both mutant and control cultures with 2 distinct centriolar spots were scored for removal of CP110 from the mother centriole. CP110 is removed in fewer mutant cells relative to control cells. *p < 0.05. (d–g) Representative images of staining for the distal appendage marker CEP164 (green) in non-ciliated (d,e) and ciliated cells (f,g). (h) Quantification of cells with CEP164-positive mother centriole or basal body. (i–l) Representative images of staining for the sub-distal appendage marker ODF2 (green) in non-ciliated (i–j) and ciliated cells (k,l). (m) Quantification of cells with ODF2-positive mother centriole or basal body. Scale bar = 5 μm. In all cases the centrosome is marked with gamma-tubulin and the axoneme with Arl13b, both in red. n.s. not significant, error bars show SEM.
Mentions: Defective axonemal extension is a common consequence of altered IFT, and accordingly C2cd3-mutant mouse cells fail to recruit a number of IFT proteins to the centrosome23. The core anterograde IFT protein IFT88 is essential for ciliogenesis and is normally localised to centrioles prior to cilia assembly, and to the transition zone and along the axoneme in ciliated cells31. Consistent with the studies in mouse cells, we observed a defect in the localisation of IFT88 at the centrioles of non-ciliated serum-starved G3P1 mutant cells (Fig. 3d–j), while all ciliated cells recruited IFT88 normally. In addition to IFT, axonemal extension is also dependent on uncapping of the distal end of the mother centriole by removal of CP1107. CP110 removal is defective in C2cd3-mutant mouse cells23, and in agreement with this we found that CP110 was not efficiently removed from the mother centriole in serum-starved G3P1 cells relative to control fibroblast cultures (23% cells in mutant cultures versus 58% in control cultures show CP110 removal; i.e CP110 at only one centriole; Fig. 4a–c). This suggests that C2CD3-mutant G3P1 cells do not efficiently uncap the mother centriole, one of the earliest steps in ciliogenesis, even when ciliogenesis is induced by serum starvation.

Bottom Line: Ciliopathies are clinically grouped in a large number of overlapping disorders, including the orofaciodigital syndromes (OFDS), the short rib polydactyly syndromes and Jeune asphyxiating thoracic dystrophy.Analysis of fibroblast cultures derived from one of these fetuses revealed a reduced ability to form cilia, consistent with previous studies in C2cd3-mutant mouse and chicken cells.More detailed analyses support a role for C2CD3 in basal body maturation; but in contrast to previous mouse studies the normal recruitment of the distal appendage protein CEP164 suggests that this protein is not sufficient for efficient basal body maturation and subsequent axonemal extension in a C2CD3-defective background.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT
Ciliopathies are a group of genetic disorders caused by defective assembly or dysfunction of the primary cilium, a microtubule-based cellular organelle that plays a key role in developmental signalling. Ciliopathies are clinically grouped in a large number of overlapping disorders, including the orofaciodigital syndromes (OFDS), the short rib polydactyly syndromes and Jeune asphyxiating thoracic dystrophy. Recently, mutations in the gene encoding the centriolar protein C2CD3 have been described in two families with a new sub-type of OFDS (OFD14), with microcephaly and cerebral malformations. Here we describe a third family with novel compound heterozygous C2CD3 mutations in two fetuses with a different clinical presentation, dominated by skeletal dysplasia with no microcephaly. Analysis of fibroblast cultures derived from one of these fetuses revealed a reduced ability to form cilia, consistent with previous studies in C2cd3-mutant mouse and chicken cells. More detailed analyses support a role for C2CD3 in basal body maturation; but in contrast to previous mouse studies the normal recruitment of the distal appendage protein CEP164 suggests that this protein is not sufficient for efficient basal body maturation and subsequent axonemal extension in a C2CD3-defective background.

No MeSH data available.


Related in: MedlinePlus