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Mutations in CDK5RAP2 cause Seckel syndrome.

Yigit G, Brown KE, Kayserili H, Pohl E, Caliebe A, Zahnleiter D, Rosser E, Bögershausen N, Uyguner ZO, Altunoglu U, Nürnberg G, Nürnberg P, Rauch A, Li Y, Thiel CT, Wollnik B - Mol Genet Genomic Med (2015)

Bottom Line: CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly.Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152.This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, University of Cologne Cologne, Germany ; Center for Molecular Medicine Cologne (CMMC), University of Cologne Cologne, Germany ; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne Cologne, Germany.

ABSTRACT
Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152.

No MeSH data available.


Related in: MedlinePlus

Characterization of CDK5RAP2-deficient Seckel fibroblasts. (A) Western blot (Wb) analysis of CDK5RAP2 expression in wild-type (control) and CDK5RAP2-deficient fibroblasts carrying the CDK5RAP2 c.4005-9A>G mutation (Seckel): Control and Seckel fibroblasts were treated with MG-132 for the indicated time or left untreated as control. The arrows indicate the position and molecular weight of wild-type CDK5RAP2 (wt) and the expected position and molecular weight of truncated CDK5RAP2 (mut). Wb analysis demonstrates the complete loss of CDK5RAP2 protein in Seckel fibroblasts. (B) Interphase and mitotic morphology of Seckel fibroblasts: Immunofluorescence staining of Seckel fibroblasts with antibodies against centrin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures I to IV), showing fragmented centrosomes (I), more centrin than pericentrin (II), multiple centrosomes (III), and misplaced centrosomes (IV). Immunofluorescence staining of Seckel fibroblasts with antibodies against alpha-tubulin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures V to IX), showing unevenly distributed cytoplasm (V), micronuclei in addition to a main nucleus (VI), failed separation of daughter cells (VII, IX), and multiple centrosomes during cell division (VIII). Scale bar, 2 μm. (C) Quantification of mitotic and centrosomal anomalies in Seckel fibroblasts. Seckel fibroblasts show abnormalities in 40% of mitoses overall compared to 9% in wt cells. The major abnormal phenotypes observed were duplicated centrosomes and misplaced centrosomes. Values are not additive. (D) DNA damage response in control and Seckel fibroblasts: Wb analysis of HU- and UV-induced phosphorylation of H2AX (Ser139) indicates that the response to DNA-damaging agents is not appreciably altered in CDK5RAP2-deficient fibroblasts.
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fig04: Characterization of CDK5RAP2-deficient Seckel fibroblasts. (A) Western blot (Wb) analysis of CDK5RAP2 expression in wild-type (control) and CDK5RAP2-deficient fibroblasts carrying the CDK5RAP2 c.4005-9A>G mutation (Seckel): Control and Seckel fibroblasts were treated with MG-132 for the indicated time or left untreated as control. The arrows indicate the position and molecular weight of wild-type CDK5RAP2 (wt) and the expected position and molecular weight of truncated CDK5RAP2 (mut). Wb analysis demonstrates the complete loss of CDK5RAP2 protein in Seckel fibroblasts. (B) Interphase and mitotic morphology of Seckel fibroblasts: Immunofluorescence staining of Seckel fibroblasts with antibodies against centrin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures I to IV), showing fragmented centrosomes (I), more centrin than pericentrin (II), multiple centrosomes (III), and misplaced centrosomes (IV). Immunofluorescence staining of Seckel fibroblasts with antibodies against alpha-tubulin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures V to IX), showing unevenly distributed cytoplasm (V), micronuclei in addition to a main nucleus (VI), failed separation of daughter cells (VII, IX), and multiple centrosomes during cell division (VIII). Scale bar, 2 μm. (C) Quantification of mitotic and centrosomal anomalies in Seckel fibroblasts. Seckel fibroblasts show abnormalities in 40% of mitoses overall compared to 9% in wt cells. The major abnormal phenotypes observed were duplicated centrosomes and misplaced centrosomes. Values are not additive. (D) DNA damage response in control and Seckel fibroblasts: Wb analysis of HU- and UV-induced phosphorylation of H2AX (Ser139) indicates that the response to DNA-damaging agents is not appreciably altered in CDK5RAP2-deficient fibroblasts.

Mentions: To investigate whether the identified c.4005-9A>G CDK5RAP2 mutation causes a complete loss of protein function, we analyzed the expression of CDK5RAP2 in primary fibroblasts established from the index patient of family SK-1 (II.1 in Fig.1A) and from an age and sex-matched healthy control individual. We confirmed the presence of alternatively spliced CDK5RAP2 mRNA in cells of the affected individual by RT-PCR (Fig.3A). In contrast, no protein expression of truncated CDK5RAP2 could be detected in Seckel fibroblasts, implying a loss of protein function (Fig.4A). Upon treatment of cells with MG-132, a potent inhibitor of the ubiquitin-dependent proteasome system, no residual mutant protein was detected, suggesting that alternative pathways are responsible for the degradation of the truncated protein.


Mutations in CDK5RAP2 cause Seckel syndrome.

Yigit G, Brown KE, Kayserili H, Pohl E, Caliebe A, Zahnleiter D, Rosser E, Bögershausen N, Uyguner ZO, Altunoglu U, Nürnberg G, Nürnberg P, Rauch A, Li Y, Thiel CT, Wollnik B - Mol Genet Genomic Med (2015)

Characterization of CDK5RAP2-deficient Seckel fibroblasts. (A) Western blot (Wb) analysis of CDK5RAP2 expression in wild-type (control) and CDK5RAP2-deficient fibroblasts carrying the CDK5RAP2 c.4005-9A>G mutation (Seckel): Control and Seckel fibroblasts were treated with MG-132 for the indicated time or left untreated as control. The arrows indicate the position and molecular weight of wild-type CDK5RAP2 (wt) and the expected position and molecular weight of truncated CDK5RAP2 (mut). Wb analysis demonstrates the complete loss of CDK5RAP2 protein in Seckel fibroblasts. (B) Interphase and mitotic morphology of Seckel fibroblasts: Immunofluorescence staining of Seckel fibroblasts with antibodies against centrin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures I to IV), showing fragmented centrosomes (I), more centrin than pericentrin (II), multiple centrosomes (III), and misplaced centrosomes (IV). Immunofluorescence staining of Seckel fibroblasts with antibodies against alpha-tubulin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures V to IX), showing unevenly distributed cytoplasm (V), micronuclei in addition to a main nucleus (VI), failed separation of daughter cells (VII, IX), and multiple centrosomes during cell division (VIII). Scale bar, 2 μm. (C) Quantification of mitotic and centrosomal anomalies in Seckel fibroblasts. Seckel fibroblasts show abnormalities in 40% of mitoses overall compared to 9% in wt cells. The major abnormal phenotypes observed were duplicated centrosomes and misplaced centrosomes. Values are not additive. (D) DNA damage response in control and Seckel fibroblasts: Wb analysis of HU- and UV-induced phosphorylation of H2AX (Ser139) indicates that the response to DNA-damaging agents is not appreciably altered in CDK5RAP2-deficient fibroblasts.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4585455&req=5

fig04: Characterization of CDK5RAP2-deficient Seckel fibroblasts. (A) Western blot (Wb) analysis of CDK5RAP2 expression in wild-type (control) and CDK5RAP2-deficient fibroblasts carrying the CDK5RAP2 c.4005-9A>G mutation (Seckel): Control and Seckel fibroblasts were treated with MG-132 for the indicated time or left untreated as control. The arrows indicate the position and molecular weight of wild-type CDK5RAP2 (wt) and the expected position and molecular weight of truncated CDK5RAP2 (mut). Wb analysis demonstrates the complete loss of CDK5RAP2 protein in Seckel fibroblasts. (B) Interphase and mitotic morphology of Seckel fibroblasts: Immunofluorescence staining of Seckel fibroblasts with antibodies against centrin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures I to IV), showing fragmented centrosomes (I), more centrin than pericentrin (II), multiple centrosomes (III), and misplaced centrosomes (IV). Immunofluorescence staining of Seckel fibroblasts with antibodies against alpha-tubulin (green) and pericentrin (red), and DAPI staining of DNA (blue) (pictures V to IX), showing unevenly distributed cytoplasm (V), micronuclei in addition to a main nucleus (VI), failed separation of daughter cells (VII, IX), and multiple centrosomes during cell division (VIII). Scale bar, 2 μm. (C) Quantification of mitotic and centrosomal anomalies in Seckel fibroblasts. Seckel fibroblasts show abnormalities in 40% of mitoses overall compared to 9% in wt cells. The major abnormal phenotypes observed were duplicated centrosomes and misplaced centrosomes. Values are not additive. (D) DNA damage response in control and Seckel fibroblasts: Wb analysis of HU- and UV-induced phosphorylation of H2AX (Ser139) indicates that the response to DNA-damaging agents is not appreciably altered in CDK5RAP2-deficient fibroblasts.
Mentions: To investigate whether the identified c.4005-9A>G CDK5RAP2 mutation causes a complete loss of protein function, we analyzed the expression of CDK5RAP2 in primary fibroblasts established from the index patient of family SK-1 (II.1 in Fig.1A) and from an age and sex-matched healthy control individual. We confirmed the presence of alternatively spliced CDK5RAP2 mRNA in cells of the affected individual by RT-PCR (Fig.3A). In contrast, no protein expression of truncated CDK5RAP2 could be detected in Seckel fibroblasts, implying a loss of protein function (Fig.4A). Upon treatment of cells with MG-132, a potent inhibitor of the ubiquitin-dependent proteasome system, no residual mutant protein was detected, suggesting that alternative pathways are responsible for the degradation of the truncated protein.

Bottom Line: CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly.Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152.This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152.

View Article: PubMed Central - PubMed

Affiliation: Institute of Human Genetics, University of Cologne Cologne, Germany ; Center for Molecular Medicine Cologne (CMMC), University of Cologne Cologne, Germany ; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne Cologne, Germany.

ABSTRACT
Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been shown to be causally involved in autosomal recessive primary microcephaly. We establish CDK5RAP2 as a disease-causing gene for Seckel syndrome and show that loss of functional CDK5RAP2 leads to severe defects in mitosis and spindle organization, resulting in cells with abnormal nuclei and centrosomal pattern, which underlines the important role of centrosomal and mitotic proteins in the pathogenesis of the disease. Additionally, we present an intriguing case of possible digenic inheritance in Seckel syndrome: A severely affected child of nonconsanguineous German parents was found to carry heterozygous mutations in CDK5RAP2 and CEP152. This finding points toward a potential additive genetic effect of mutations in CDK5RAP2 and CEP152.

No MeSH data available.


Related in: MedlinePlus