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Nat Genet. Author manuscript; available in PMC Aug 30, 2012.
Published in final edited form as:
Published online Dec 5, 2010. doi:  10.1038/ng.725
PMCID: PMC3430850

CEP152 is a genome maintenance protein disrupted in Seckel syndrome


Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation.

Maintenance of genomic integrity is required for cell survival and function. As the accumulation of DNA damage has an important effect on cell viability, organisms have evolved mechanisms to protect the integrity of DNA by inducing DNA damage responses and cell cycle arrest or by triggering apoptosis1. Disruption of these mechanisms can lead to increased genomic instability, which is a key factor for the development of cancer and is involved in aging. Two important regulators of DNA-damage responses are the phosphoinositide 3-kinase–related serine/threonine kinases ATM (Ataxia-Telangiectasia Mutated) and ATR (Ataxia-Telangiectasia and Rad3 related)1,2. Whereas ATM is activated in response to DNA double-strand breaks, ATR is involved mainly in responses to single-stranded DNA induced by ultraviolet light damage or replication arrest. ATM and ATR both trigger an overlapping set of cellular responses that promote cell cycle arrest and DNA repair3.

Recently, it was reported that a hypomorphic ATR mutation is associated with embryonic replicative stress, accelerated aging in mice and Seckel syndrome in humans4,5. Seckel syndrome (MIM210600) is a heterogeneous autosomal recessive disorder characterized by a proportionate short stature, severe microcephaly and mental retardation, and a typical ‘bird-head’ facial appearance6. Initially, we clinically evaluated five consanguineous families with Seckel syndrome originating from an isolated rural area in Turkey (Fig. 1, Supplementary Figs. 1 and 2 and Supplementary Table 1). We genotyped DNA from four affected members of three families using the 250K SNP Array (Affymetrix) and obtained a single maximum log10 odds (LOD) score of 6.03 for a region between rs1598206 and rs2330591 on chromosome 15q21.1–q21.2 (Fig. 1b). Subsequent fine mapping using microsatellite markers confirmed shared homozygosity and a founder haplotype in a 3.4-Mb region between markers D15S123 and D15S1017. This genomic region contains 28 known and predicted genes. Microcephalic osteodysplastic primordial dwarfism type II (MOPDII, MIM210720), which shows overlapping features with Seckel syndrome, was recently associated with mutations in PCNT, the centrosomal pericentrin gene7,8. Hence, we considered CEP152, the centrosomal protein 152 gene located in the critical region, to be a highly relevant candidate gene.

Figure 1
Clinical and molecular characterization of CEP152 Seckel subjects. (a) Clinical characteristics of subjects 442, 443 and 586 presenting with microcephaly, sloping forehead, high nasal bridge, beaked nose and retrognathia. Informed consents to publish ...

Sequencing of all 27 exons of CEP152 (Supplementary Table 4) revealed a homozygous splice donor-site mutation in intron 4, c.261+1G>C, which co-segregated with the founder haplotype and the disease in all affected family members (Supplementary Fig. 3) and was not found in 250 healthy Turkish control individuals. The c.261+1G>C mutation completely disrupted the splice donor site, as shown through RT-PCR analysis of RNA from affected individuals. We found four different aberrant transcripts likely to cause loss of protein function though partial functional activity of one mutant protein, Val86_Asn87del, could not be excluded (Supplementary Fig. 4).

Independently, we identified CEP152 as the causative gene in a French individual with Seckel syndrome of Turkish origin born to consanguineous parents through the use of an exome sequencing strategy (Supplementary Methods). We identified seven new and homozygous nonsense and essential splice-site variants that were expected to have a severe impact on gene function (Supplementary Table 2). Of these seven variants, a G>C transversion at the +1 position of a donor site was embedded within a ~35-Mb tract of homozygosity on chromosome 15, which was the longest tract of homozygosity observed on any autosome in this individual (Fig. 1c). None of the other variants were associated with a large region of homozygosity. This mutation is the same c.261+1G>C Turkish founder mutation in CEP152 described above. These data demonstrated that mutation identification through exome sequencing, in conjunction with simultaneous analysis of homozygous stretches, can be used as an efficient approach to gene identification in autosomal recessive disorders.

In addition, sequencing CEP152 in further subjects with Seckel syndrome identified two individuals that were compound heterozygous for likely loss-of-function mutations. One subject of Italian origin from Germany was compound heterozygous for a nonsense mutation, c.2034T>G (p.Tyr678X), and an intron 19-splice donor-site mutation, c.2694+1G>T, leading to retention of the entire intron 19 in the CEP152 mRNA (r.2694G_ins3581, Ile899LeufsX29) (Supplementary Fig. 5). Because the phenotype of this subject was as severe as the phenotypes seen in the Turkish families, it is likely that the c.261+1G>C Turkish founder mutation has similar functional consequences as the mutation seen here. A subject from South Africa was compound heterozygous for a paternally inherited 2-bp deletion, c.4210–4211delGT (p.Val1404fsX2, exon 27), and a maternally inherited missense mutation, c.2000A>G (exon 15), affecting the highly conserved lysine at position 667 (p.Lys667Arg) (Fig. 1d and Supplementary Fig. 3). Very recently, mutations in CEP152 have been associated with microcephaly in an Eastern Canadian subpopulation9.

CEP152 encodes a 1,654 amino acid protein that was originally identified in a proteomic screen of human centrosomes10. Analysis of the subcellular localization of asl (asterless), the Drosophila ortholog of CEP152, has shown it to be associated with the periphery of centrioles, where it is involved in the initiation of centriole duplication11. Expression of CEP152 in HEK293T cells revealed fluorescence staining of the centrosomes, where it co-localized with pericentrin (Fig. 2)12. To determine the functional effect of CEP152 deficiency on genomic stability, we analyzed the morphology of CEP152-deficient Seckel fibroblasts during interphase and during different stages of mitosis. A substantial number of Seckel fibroblasts contained multiple, differently sized nuclei and centrosomes, micronuclei and fragmented centrosomes during interphase (Fig. 2a and Supplementary Fig. 6). Metaphase karyotyping of CEP152-deficient Seckel lymphocytes showed aneuploidy in 15 out of a total 109 metaphase spreads (Fig. 2b). During metaphase, we observed incorrectly aligned chromosomes, monopolar spindles with a single large centrosome, triple spindles with differently sized and structurally compromised centrosomes, and prematurely separated sister chromatids in Seckel cells. Statistical analysis revealed that CEP152 deficiency leads to an increased number of cells containing multiple nuclei and centrosomes, fragmented centrosomes and an increased frequency of aberrant cell divisions.

Figure 2
Characterization of CEP152 Seckel cells. (a) Mitotic morphology of CEP152 Seckel fibroblasts. Immunofluorescence staining of CEP152 Seckel fibroblasts carrying the c.261+1G>C mutation with antibodies against α-tubulin (green), pericentrin ...

Moreover, the number of CEP152-deficient cells in telophase was decreased. Most strikingly, CEP152-deficient cells appeared to be arrested at early anaphase, reflected by an increased number of early anaphase figures in fixed Seckel populations when compared to the wildtype population (Supplementary Fig. 6f). This block may have resulted from problems with chromatid alignment, uneven pulling forces in the spindle, or activation of a checkpoint that responds to weakly attached or misaligned chromosomes. There did not appear to be additional apoptosis at early anaphase. Taken together, these data clearly showed centrosomal and mitotic aberrations caused by CEP152 mutations.

In addition, Seckel cells showed an overall increased sensitivity to oxidative stress and responded to this stress with increased apoptosis (Supplementary Fig. 7). Cell cycle analysis in CEP152 knockdown cells using short hairpin RNA suggested that CEP152 deficiency delays S-phase entry. Furthermore, fewer Seckel cells progressed to the G2/M phase and an increased proportion of Seckel cells stayed in G0/G1. These findings suggest altered ATR-mediated checkpoint activity and increased replicative stress in CEP152-deficient cells.

In order to determine possible defects in DNA repair mechanisms, we measured the mitomycin-C–induced sister chromatid exchange frequency. We observed a substantial increase in chromosome instability in CEP152-deficient lymphocytes (Supplementary Table 3). In a yeast two-hybrid (Y2H) screen using an N-terminal deletion construct of CEP152 as bait, the CDK2-interacting protein (CINP) was identified as an interaction partner of CEP152 in three independent hits. We confirmed the CEP152-CINP interaction by using complementary immunoprecipitation approaches, and we showed that CEP152 constitutively binds to CINP (Supplementary Fig. 8). We observed a weak nuclear staining for both CEP152 and CINP (data not shown). CINP has recently been identified as a genome maintenance protein13 that interacts with the ATR-interacting protein (ATRIP) and has an important function in ATR-mediated checkpoint signaling. Silencing of CINP causes increased γH2AX foci formation, suggesting that CINP contributes to the formation of a functional unit in DNA damage response. The identification of CEP152 as a binding partner of CINP provides additional evidence for an important role of CEP152 in DNA-damage response and genome maintenance.

ATR-mediated phosphorylation of CHK1 and H2AX are the initial steps in response to DNA damage and increased replicative stress14,15. We did not observe a detectable alteration in hydroxyurea-induced phosphorylation of CHK1 in CEP152-deficient cells (Fig. 2d). However, we found that replicative stress leads to enhanced CHK2 phosphorylation in Seckel cells (Supplementary Fig. 8), indicating an increased activation of ATM signaling pathways. Furthermore, we found that formation of hydroxyurea-induced γH2AX foci in CEP152-deficient cells was increased. Protein blot analysis confirmed that the levels of γH2AX were considerably higher in hydroxyurea-treated CEP152-deficient cells as compared to wildtype cells. There are likely to be additional mechanisms by which CEP152 might affect ATR checkpoint control and subsequently lead to increased replicative stress, which causes a compensatory activation of CHK2 and γH2AX. Recent analysis of H2AX phosphorylation in the ATRs/s Seckel mouse model indicated that γH2AX has been substantially increased throughout embryonic development, particularly in tissues with a high mitotic index5. In agreement with our results, a previous study suggested that accumulated replicative stress can activate ATM-dependent DNA damage response and thereby lead to increased H2AX phosphorylation5.

In conclusion, we identified the centrosomal protein CEP152 as a new protein involved in the maintenance of genomic integrity and in the ability to respond to DNA damage. Impaired CEP152 function leads to genomic instability and increased H2AX phosphorylation, a measure of accumulated replicative stress. Our data further support the essential link between genomic instability, as seen in individuals with Seckel syndrome, and the activation of histone H2AX as response to increased replicative stress.

Supplementary Material

Supplementary Figures 1-8, Supplementary Tables 1-4 and Supplementary Methods


We thank all family members who participated in this study, E. Milz, A. Alver and A. Coffey for excellent technical assistance, F. Kokocinski and A. Palotie for experimental design, and C. Kubisch, B. Wirth and K. Boss for critical reading the manuscript. The CINP antibody was a kind gift from D. Cortez (Nashville, Tennessee, USA). This work was supported by the German Federal Ministry of Education and Research (BMBF) by grant numbers 01GM0880 (SKELNET) and 01GM0801 (E-RARE network CRANIRARE) to B.W., the Karadeniz Technical University Research Fund by grant numbers 2008.114.001.02 and 2008.114.001.12 to E.K., the Medical Research Council and Lister Institute to A.P.J. and by the Wellcome Trust, grant 077014/Z/05/Z.


Accession codes. The CEP152 reference sequence is deposited in the NCBI nucleotide database under the accession code NM_014985.2.

Note: Supplementary information is available on the Nature Genetics website.

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/.


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