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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

Ciliogenesis and IFT protein recruitment is impaired in fibroblasts isolated from G3P1.(a,b) Images showing a lower percentage of cilia (as marked by Arl13b staining) relative to DAPI-stained nuclei in serum-starved C2CD3-mutant fibroblast cultures (G3P1), compared to control juvenile dermal fibroblasts. Scale bar = 10 μm. Quantification is shown in (c). (d–i) Representative images of IFT88 staining (green) in non-ciliated cells (indicated by lack of red Arl13b staining along the axoneme) in serum-starved cultures. IFT88 localises to the centrosome (stained with gamma-tubulin in red) in control fibroblasts (d–f) whereas in fibroblasts isolated from G3P1, IFT88 is absent from most centrosomes (g–i). Scale bar = 5 μm. (j) Quantification of percentage of non-ciliated cells with IFT88-postive centrosomes in cultured C2CD3-mutant fibroblasts, relative to normal juvenile fibroblasts. ***p < 0.0001, error bars show SEM. All ciliated cells show normal localisation of IFT88 in both control and mutant cultures, reflecting the requirement for IFT88 in ciliogenesis.
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f3: Ciliogenesis and IFT protein recruitment is impaired in fibroblasts isolated from G3P1.(a,b) Images showing a lower percentage of cilia (as marked by Arl13b staining) relative to DAPI-stained nuclei in serum-starved C2CD3-mutant fibroblast cultures (G3P1), compared to control juvenile dermal fibroblasts. Scale bar = 10 μm. Quantification is shown in (c). (d–i) Representative images of IFT88 staining (green) in non-ciliated cells (indicated by lack of red Arl13b staining along the axoneme) in serum-starved cultures. IFT88 localises to the centrosome (stained with gamma-tubulin in red) in control fibroblasts (d–f) whereas in fibroblasts isolated from G3P1, IFT88 is absent from most centrosomes (g–i). Scale bar = 5 μm. (j) Quantification of percentage of non-ciliated cells with IFT88-postive centrosomes in cultured C2CD3-mutant fibroblasts, relative to normal juvenile fibroblasts. ***p < 0.0001, error bars show SEM. All ciliated cells show normal localisation of IFT88 in both control and mutant cultures, reflecting the requirement for IFT88 in ciliogenesis.

Mentions: While the effect of C2CD3 mutations has previously been investigated in mouse and chicken cells202124, direct analysis of C2CD3-mutant human cells has not been reported. We had access to human fibroblasts from individual G3P1, and investigated their ability to ciliate following serum starvation to induce ciliogenesis. We found a significant reduction in the percentage of G3P1 cells with a cilium when compared to control juvenile dermal fibroblasts, following staining of the axoneme with either an Arf-like small GTPase ARL13b (Fig. 3a–c) or acetylated α-tubulin antibody (not shown). These data are consistent with previous studies in mouse21 and chicken cells24. Note that we did not have access to a normal fetal fibroblast control but did confirm that the level of ciliogenesis in a fibroblast line derived from a 14-week gestation fetus with an unrelated non-ciliopathy disease (fetal akinesia), is equivalent to that in the juvenile fibroblast line used as a control in these studies (see Supplementary Fig. S1). This suggests that ciliogenesis in fibroblasts is not affected by the age at which they are derived.


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)

Ciliogenesis and IFT protein recruitment is impaired in fibroblasts isolated from G3P1.(a,b) Images showing a lower percentage of cilia (as marked by Arl13b staining) relative to DAPI-stained nuclei in serum-starved C2CD3-mutant fibroblast cultures (G3P1), compared to control juvenile dermal fibroblasts. Scale bar = 10 μm. Quantification is shown in (c). (d–i) Representative images of IFT88 staining (green) in non-ciliated cells (indicated by lack of red Arl13b staining along the axoneme) in serum-starved cultures. IFT88 localises to the centrosome (stained with gamma-tubulin in red) in control fibroblasts (d–f) whereas in fibroblasts isolated from G3P1, IFT88 is absent from most centrosomes (g–i). Scale bar = 5 μm. (j) Quantification of percentage of non-ciliated cells with IFT88-postive centrosomes in cultured C2CD3-mutant fibroblasts, relative to normal juvenile fibroblasts. ***p < 0.0001, error bars show SEM. All ciliated cells show normal localisation of IFT88 in both control and mutant cultures, reflecting the requirement for IFT88 in ciliogenesis.
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Related In: Results  -  Collection

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f3: Ciliogenesis and IFT protein recruitment is impaired in fibroblasts isolated from G3P1.(a,b) Images showing a lower percentage of cilia (as marked by Arl13b staining) relative to DAPI-stained nuclei in serum-starved C2CD3-mutant fibroblast cultures (G3P1), compared to control juvenile dermal fibroblasts. Scale bar = 10 μm. Quantification is shown in (c). (d–i) Representative images of IFT88 staining (green) in non-ciliated cells (indicated by lack of red Arl13b staining along the axoneme) in serum-starved cultures. IFT88 localises to the centrosome (stained with gamma-tubulin in red) in control fibroblasts (d–f) whereas in fibroblasts isolated from G3P1, IFT88 is absent from most centrosomes (g–i). Scale bar = 5 μm. (j) Quantification of percentage of non-ciliated cells with IFT88-postive centrosomes in cultured C2CD3-mutant fibroblasts, relative to normal juvenile fibroblasts. ***p < 0.0001, error bars show SEM. All ciliated cells show normal localisation of IFT88 in both control and mutant cultures, reflecting the requirement for IFT88 in ciliogenesis.
Mentions: While the effect of C2CD3 mutations has previously been investigated in mouse and chicken cells202124, direct analysis of C2CD3-mutant human cells has not been reported. We had access to human fibroblasts from individual G3P1, and investigated their ability to ciliate following serum starvation to induce ciliogenesis. We found a significant reduction in the percentage of G3P1 cells with a cilium when compared to control juvenile dermal fibroblasts, following staining of the axoneme with either an Arf-like small GTPase ARL13b (Fig. 3a–c) or acetylated α-tubulin antibody (not shown). These data are consistent with previous studies in mouse21 and chicken cells24. Note that we did not have access to a normal fetal fibroblast control but did confirm that the level of ciliogenesis in a fibroblast line derived from a 14-week gestation fetus with an unrelated non-ciliopathy disease (fetal akinesia), is equivalent to that in the juvenile fibroblast line used as a control in these studies (see Supplementary Fig. S1). This suggests that ciliogenesis in fibroblasts is not affected by the age at which they are derived.

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