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A Truncating of CEP135 Causes Primary and Disturbed Centrosomal Function

Muhammad Sajid Hussain,1,2,3,4 Shahid Mahmood Baig,4 Sascha Neumann,2 Gudrun Nu¨rnberg,1,3 Muhammad Farooq,4 Ilyas Ahmad,1,4 Thomas Alef,1 Hans Christian Hennies,1,5 Martin Technau,2 Janine Altmu¨ller,1 Peter Frommolt,1,5 Holger Thiele,1 Angelika Anna Noegel,1,2,3,5,* and Peter Nu¨rnberg1,3,5,*

Autosomal-recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by intellectual disability, reduced brain and head size, but usually without defects in cerebral cortical architecture, and other syndromic abnormalities. MCPH is heterogeneous. The underlying of the seven known loci code for centrosomal . We studied a family from northern Pakistan with two microcephalic children using homozygosity mapping and found suggestive linkage for regions on 2, 4, and 9. We sequenced two positional candidate genes and identified a homozygous frameshift mutation in the encoding the 135 kDa centro- somal (CEP135), located in the linkage interval on 4, in both affected children. Post hoc whole-exome sequencing corroborated this mutation’s identification as the causal variant. Fibroblasts obtained from one of the patients showed multiple and frag- mented , disorganized , and reduced growth rate. Similar effects were reported after knockdown of CEP135 through RNA interference; we could provoke them also by ectopic overexpression of the protein. Our findings suggest an addi- tional for MCPH at HSA 4q12 (MCPH8), further strengthen the role of centrosomes in the development of MCPH, and place CEP135 among the essential components of this important organelle in particular for a normal neurogenesis.

Autosomal-recessive primary microcephaly (MCPH [MIM organization at the , resulting in altered 251200]) is a neurodevelopmental disorder characterized division and cortical development. Despite their similar by reduced size of the and mild to moderate subcellular localization, the biological functions of these intellectual disability whereas the architecture of the brain genes may vary.1,3,4,10 is largely normal. Head circumference is already reduced at We studied a consanguineous family from northern birth and usually more than 3 SD below the age- and sex- Pakistan; the two affected children had primary micro- matched population mean throughout the patient’s life- cephaly at birth (Figures 1A and 1B). After informed time. Many patients have a receding forehead. MCPH is consent of the parents was obtained, pedigree information heterogeneous; it has seven known loci, MCPH1– was documented and clinical data and blood samples were MCPH7,1 each of which is associated with an underlying collected from the affected siblings, their father, and an genetic defect: MCPH1 (MIM 251200) is associated with unaffected sibling. Each affected child had a sloping fore- of MCPH1 (MIM 607117);2 MCPH2 with or head. By 5 years of age they showed severe cognitive defi- without cortical malformations (MIM 604317) is associ- cits; their speech was not understandable. They were ated with mutations of WDR62 (MIM 613583);3,4 unable to form sentences and even to say any clear words, MCPH3 (MIM 604804) is associated with mutations of although their hearing was not impaired. Other abnormal- CDK5RAP2 (MIM 608201);5 MCPH4 (MIM 604321) is asso- ities were not apparent. Individual V-2 died at 11 years. The ciated with mutations of CEP152 (MIM 613529);6 MCPH5 head circumference of the affected individuals ranged (MIM 608716) is associated with mutations of ASPM (MIM between 12 and 14.5 SD compared to the average pop- 605481);7 MCPH6 (MIM 608393) is associated with muta- ulation of the same age and sex. The parents were healthy tions of CENPJ (MIM 609279);5 and MCPH7 (MIM 612703) with normal head circumference. Ethical approval for this is associated with mutations of STIL (MIM 181590).8 study was obtained from the ethics review board at the Recently, a new locus at HSA 10q11.23-21.3 was described National Institute for Biotechnology and Genetic Engi- in a consanguineous Turkish family, but the authors were neering in Faisalabad according to the Declaration of Hel- not able to find the disease-causing gene variant.9 The sinki. MCPH-associated genes described to date have been impli- Standard procedures were used to extract DNA from the cated in cell division and regulation, and many of blood samples for homozygosity mapping. Initially, we the corresponding gene products are localized to the excluded all known MCPH loci in the family, using STR centrosome. It has been speculated that they affect neural markers to demonstrate heterozygosity in the relevant progenitor cell number through disturbed genomic regions. We then performed a -wide

1Cologne Center for Genomics (CCG), University of Cologne, 50931 Cologne, Germany; 2Institute of Biochemistry I, Medical Faculty, University of Cologne, 50931 Cologne, Germany; 3Center for Molecular Medicine Cologne (CMMC), University of Cologne, 50931 Cologne, Germany; 4Health Biotech- nology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad 38000, Pakistan; 5Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50674 Cologne, Germany *Correspondence: [email protected] (A.A.N.), [email protected] (P.N.) DOI 10.1016/j.ajhg.2012.03.016. Ó2012 by The American Society of Genetics. All rights reserved.

The American Journal of 90, 871–878, May 4, 2012 871 Figure 1. Identification of an MCPH-Causing Mutation in CEP135 (A) Clinical features of two siblings of a family from northern Pakistan presenting with microcephaly, sloping forehead, and retrogna- thia. Informed consent to publish the photographs was obtained from the subjects’ parents. (B) A simplified pedigree of the Pakistani family. Filled circles indicate individuals with MCPH. Their parents are second cousins. Only the core family is shown. DNA was available for individuals IV-1, V-1, V-2, and V-3. (C) G-banded showing the position of the linkage interval at 4p14-4q12 along with the position of CEP135. The homo- zygous region was delimited by the markers SNP_A-2154951 (rs12498424, physical position 40,631,476 bp) and SNP_A-1894332 (rs13134527, physical position 58,702,130 bp). (D) Sequence chromatograms of a part of CEP135 exon 8 as obtained by Sanger sequencing. Traces of the male patient (V-1) and his father (IV-1) show the mutation c.970delC in homozygous and heterozygous status, respectively. The mutant sequences are shown along with a wild-type trace from a control individual. (E) Schematic representation of the genomic structure of human CEP135. The 26 exons of CEP135 are drawn to scale, while introns show just artificial lines. Black boxes represent untranslated regions. The position of mutation c.970delC in exon 8 is indicated. (F) CEP135 structure as predicted by SMART (Simple Modular Architecture Research Tool) database. This software predicted six coiled- coil domains (orange rods) which cover almost the entire region of the protein. The position of mutation p.Q324Sfs*2 in the fourth coiled-coil domain is indicated. linkage analysis using the Affymetrix GeneChip Human hg19). The genes were prioritized with Endeavour and Mapping 250K Sty Array. Data handling, evaluation, and GeneWanderer.12,13 Highly ranked genes were further statistical analysis were performed as described previ- scrutinized manually with the NCBI, Ensembl, and UCSC ously.11 We observed three peaks on chromosomes 2, 4, genome databases. Finally, we gave top priority to genes and 9 that were suggestive for linkage with maximum mul- that were very likely to have important functions related tipoint LOD scores of 2.07, 2.53, and 2.53, respectively to cell division or chromosome segregation and decided (Figure S1). The underlying common homozygous regions to sequence the following two strong candidate genes defined a candidate region of 8.6 Mb on chromosome first: the cell division cycle-14 homolog B gene (CDC14B 2 (90,183,484–98,795,792; hg19), a candidate region of [MIM 603505]) at cytoband 9q22.32-q31.1 and the gene 18.1 Mb on chromosome 4 (40,631,476–58,702,130; encoding the 135 kDa centrosomal protein (CEP135 hg19), and a candidate region of 8.8 Mb on chromosome [MIM 611423]) at HAS 4p14-q12 (Figure 1C). 9 (92,274,161–101,122,314; hg19). Altogether these All exons and the intron-exon boundaries of CEP135 regions include more than 200 annotated known and and CDC14B were sequenced in the two affected individ- predicted coding genes (UCSC Genome Bioinformatics, uals. The PCR products (primers are listed in Table S1)

872 The American Journal of Human Genetics 90, 871–878, May 4, 2012 were sequenced bidirectionally with a BigDye Terminator Project, Seattle) (Table S3). Only three other homozygous v1.1 cycle sequencing on an ABI3730xl automated variants resisted our filter criteria in addition to CEP135 DNA sequencer. Sequences were analyzed with DNASTAR c.970delC. None of these could be assumed to be relevant (Lasergene) and Mutation Surveyor (SoftGenetics). We for the phenotype (Table S4). Likewise, we could not find found no mutation in CDC14B but a homozygous single deleterious mutations in any of the seven known MCPH- base-pair (bp) (c.970delC) in exon 8 of CEP135. associated genes, even when allowing for compound We found the homozygous 1 bp deletion in both affected heterozygosity during filtering. children, but not in the unaffected child, whereas the The deletion c.970delC of CEP135 results in a frameshift, father was heterozygous for this mutation (Figure 1D), changing glutamine at position 324 into serine, immedi- which is compatible with recessive inheritance. Moreover, ately followed by a premature termination codon the mutation is unlikely to be a polymorphism, as it is not (p.Gln324Serfs*2). This truncating mutation is obviously listed in dbSNP, and we could not find it when testing 384 incompatible with a normal function of CEP135 if one healthy Pakistani controls with pyrosequencing. For this considers the size of the protein and the position of purpose, PCR primers (listed in Table S2) were designed the mutation (Figures 1E and 1F). CEP135 consists of 26 by the PSQ Assay Design program v.1.0.6 (QIAGEN, exons, and its open reading frame codes for a polypeptide Hilden, Germany). Pyrosequencing was done according of 1,140 amino acids. to the manufacturer’s instructions on a PSQ HS96A instru- CEP135 is a conserved a-helical protein which is present ment (QIAGEN) with the use of PyroMark Gold Q96 at the centrosome throughout the cell cycle. Electron- Reagents (QIAGEN). The data were analyzed by Pyro microscopic studies showed its association with the Q-CpG v.1.0.9 analysis software (QIAGEN). , an electron-dense material sur- In an attempt to identify a second independent muta- rounding the . Reducing CEP135 amounts in tion of CEP135, we sequenced CEP135 in patients from cells via RNA interference caused a disorganization of seven other families affected with MCPH from northern interphase and mitotic spindles, leading to the hypothesis Pakistan in which all known MCPH-associated genes that CEP135 has a role in maintaining the structure and were previously excluded. No CEP135 mutation was found organization of the centrosome and of microtubules.15 in any of these families. Therefore, we decided to perform More recently the protein was identified as a centriolar whole-exome sequencing of the affected boy (individual component; it functions in biogenesis and V-1) of the family presented in Figure 1B in order to presumably has a scaffolding role.16 Centrioles are core demonstrate that there were no other mutations in rele- components of animal centrosomes and act as basal bodies vant genes that might also explain the phenotype. We to assemble cilia and flagella.17 fragmented 1 mg of DNA using sonification technology To unravel the effect of the mutation on the cellular (Covaris, Woburn, MA, USA). The fragments were end re- level, we analyzed control and patient fibroblasts. Biopsies paired and adaptor ligated. After size selection, the library were taken from the affected individual V-1 (Figure 1B) and was subjected to the enrichment process. We chose the Seq- healthy individuals. Tissues were cleaned with antiseptic Cap EZ Human Exome Library v2.0 kit from NimbleGen agent (Betaisodona, Mundipharma) and incubated over- (Roche NimbleGen, Madison, WI, USA) and analyzed the night with Dispase II (1.5 U/ml, Roche) diluted in PBS, sample on an Illumina HiSeq 2000 sequencing instrument. pH 6.8, at 4C to separate the intact epidermis from the About 10 Gb of sequence were produced for this sample by dermis. The dermis was incubated in Dulbecco’s modified loading it individually on one lane of a flow cell and gener- Eagle’s medium (DMEM) at 37C. After one week, fibro- ating paired-end reads of 2 3 100 bp. This resulted in a very blasts were detected. The pool of fibroblasts was increased high coverage; i.e., > 303 for nearly 92% of the target by additional culturing. Primary fibroblasts established sequences, which comprised about 44 Mb. Primary data from the patient grew very slowly. To enhance growth, were filtered according to signal purity by the Illumina DMEM with 15% fetal bovine serum was used. Patient Realtime Analysis (RTA) software v1.8. Subsequently, the and wild-type primary fibroblasts were then cultured on reads were mapped to the reference build 12 mm coverslips and fixed with 3% paraformaldehyde. hg19 via the ELANDv2 alignment algorithm on a multi- For staining of microtubules, cells were incubated for node compute cluster. With the use of CASAVA v1.8, PCR 15 min in stabilization buffer, which is composed duplicates were filtered out, and the output was converted of Hank’s buffer (137 mM NaCl, 5 mM KCl, 1.1 mM into BAM format. Variant calling was performed with the Na2HPO4, 0.4 mM KH2PO4, 5 mM Glucose, 4 mM 14 use of SAMtools (version 0.1.7) for indel detection. NaHCO3) containing 1 mM MES, pH 6.8, 2 mM EGTA,

Scripts developed in-house at the Cologne Center for and 2 mM MgCl2. Permeabilization was done by 0.5% Genomics were applied to detect protein changes, affected Triton X-100 in 13 tubulin stabilization buffer for 4 min splice sites, and overlaps with known variants. In partic- at room temperature. For g-tubulin detection, the cells ular, we filtered the variants for high-quality unknown were fixed with prechilled methanol for 10 min at 20C. variants in the linkage intervals (dbSNP build 132 or the Subsequently, the fixed cells were treated three times with 1000 database; in-house variation database; and tubulin stabilization buffer. Blocking was done for 15 min public Exome Variant Server, NHLBI Exome Sequencing with blocking buffer (13 PBG: PBS containing 5% BSA and

The American Journal of Human Genetics 90, 871–878, May 4, 2012 873 Figure 2. Centrosome, Microtubule, and Nuclear Shape Defects in CEP135 Microce- phalic Patient Cells (A) Immunofluorescence staining and confocal microscopy images of wild-type primary fibroblasts with well-organized microtubules and a single centrosome de- tected by g-tubulin. g-tubulin (turquoise) and a-tubulin (green) antibodies were used. DAPI (blue) was used for DNA stain- ing. Scale bar, 5 mm. (B) Abnormality of centrosome number in CEP135 patient primary fibroblasts. Interphase cell showing supernumerary centrosomes. In this figure three centro- somes were observed as detected by g-tubulin (turquoise). Scale bar, 5 mm. (C) Disorganized microtubule network in patient fibroblasts. Scale bar, 20 mm. (D) Control primary fibroblast showing a well-shaped ellipsoid nucleus. Scale bar, 5 mm. (E) Mutant fibroblast with a dysmorphic nucleus. Scale bar, 10 mm. (F) Graphical representation showing the number of CEP135 mutant primary fibroblasts with dysmorphic nuclei. About 20% of mutant fibroblasts harbored misshapen nuclei whereas in control fibro- blasts this number was only ~3%. Three hundred cells of each, wild-type and mutant, were counted. Error bars represent SEM, p ¼ 3.44 3 10 03 (Student’s t test).

the vicinity of the nucleus during interphase (Figure 2A). In the patient’s primary fibroblasts, the centrosome number was increased in more than 18% of the cells (Table S5). In such cells we found 3, 4, or 5 centrosomes per cell (Figures 2B and S2). Furthermore, centrosomes 0.45% fish gelatin). Primary antibodies were diluted in appeared fragmented (Figure S2). The microtubule network blocking buffer and incubated overnight at 4C. The was frequently disorganized (~55% of the cells), which was following antibodies were used: mouse monoclonal anti accompanied by cell shape changes (Figures 2C and S3; g-tubulin (Sigma-Aldrich, GTU-88; 1:300), rabbit poly- Table S5). We also observed misshapen and fragmented clonal anti-pericentrin (Abcam, ab4448; 1:300), and rat nuclei (Figure 2E and S4). Statistical analysis showed monoclonal anti a-tubulin (YL 1/2 1:20).18 After incuba- that ~20% of the mutant cells harbored misshapen nuclei tion, samples were treated with 13 PBS three times for as compared to ~3% in control fibroblasts (Figure 2F; Table 5 min followed by secondary antibody incubation S5). Another prominent aspect of the patient’s primary (1:1,000 diluted in blocking buffer) for one hour at room fibroblasts was the complete loss of centrosomes. Approx- temperature. Alexa Fluor 568 goat anti-mouse IgG (Invitro- imately 22% of mutant primary fibroblasts were without gen, A11004), Alexa Fluor 647 donkey anti-mouse IgG centrosomes as detected with g-tubulin, whereas this was (Invitrogen, A31571), and Alexa Fluor 488 goat anti-rat never observed in control cells (Table S5; Figure S3). Most IgG (Invitrogen, A11006) were used as secondary anti- cells with misshapen nuclei were also devoid of centro- bodies. DNA was detected with DAPI (Sigma-Aldrich, somes (Figure S4). D9564). Finally, the cells were mounted on glass slides For ectopic expression of wild-type and mutant with Gelvatol. Images were taken with a confocal micro- (c.970delC) CEP135 fused to green fluorescent protein scope (Leica, LSM TCS SP5). (GFP), we used COS-7 cells. Gateway Technology (Invitro- Control primary fibroblasts had oval nuclei surrounded gen) was employed to clone wild-type (NM_025009.3) by organized microtubules and a single centrosome in CEP135 cDNA. The CEP135 mutation (c.970delC) was

874 The American Journal of Human Genetics 90, 871–878, May 4, 2012 Figure 3. Distribution of GFP-Tagged Wild-Type and Mutant CEP135 (A) N-terminally GFP-tagged human CEP135 (GFP-CEP135) was transiently expressed in COS-7 cells. GFP-CEP135 is present in dots of variable size and number. A single centrosome was detected by pericentrin-specific antibodies (tur- quoise). Scale bar, 5 mm. (B) COS-7 cell transfected with GFP-tagged mutant CEP135 (GFP-CEP135-mut), where diffuse staining was detected. Scale bar, 5 mm. (C) Abnormal microtubule network in COS-7 expressing wild-type CEP135. Scale bar, 5 mm. (D) COS-7 cells expressing GFP-CEP135- mut have a disorganized microtubule network. Microtubules appeared broken and concentrated near the nucleus. Scale bar, 5 mm.

expression of both wild-type and mutant GFP-tagged CEP135 in HaCaT cells led to the presence of abnormal microtubule networks. The severity of the disorganization was more pronounced in cells expressing the mutant protein (Figure 3C and 3D). Cells transfected with GFP-tagged mutant CEP135 also harbored mul- tiple centrosomes (up to 5), which then resulted in multipolar spindle formation. This phenotype was not observed in the cells that overex- pressed the wild-type protein. Inter- estingly, the GFP-tagged CEP135 was detected on microtubules (Figure 4). These findings of a disorganized introduced into the full-length cDNA with the use of the microtubule network and multiple centrosomes in cells QuikChange II Site-Directed Mutagenesis Kit (Stratagene) transfected with GFP-tagged CEP135 resembled those (primers are listed in Table S6) in an attempt to reproduce observed in mutant primary fibroblast cells. the patient’s situation. Entry clones in pENTR/TEV/ The CEP135 mutation c.970delC is thought to lead to a D-TOPO were transformed into Gateway destination truncation of the protein due to the introduction of a vector pcDNA-DEST53, which has an N-terminal cycle-3 premature stop codon (p.Gln324Serfs*2). Alternatively, GFP tag. Both wild-type and mutant (10 mg/ml) it might also trigger nonsense-mediated mRNA decay were used for transfection of COS-7 cells. The Gene Pulser (NMD).19 We designed RT-PCR experiments to investigate II (Bio-Rad) device was used for electroporation. 72 hr this (Table S7). The PAXgene Blood RNA system (QIAGEN) after transfection, the cells were subjected to immuno- was used to extract RNA from patient and control blood fluorescence. samples, and cDNA was synthesized with SuperScript III When we analyzed the localization of the proteins and reverse transcriptase (Invitrogen). When primers their effect on centrosomes and microtubule organization, were used to amplify a nearly full-length CEP135 cDNA we observed wild-type GFP-tagged CEP135 at the centro- of ~3.4 kb, no PCR product was obtained with the patient’s some where it colocalized with the centrosomal protein RNA, but only with the control sample, suggesting pericentrin. We also detected it in some spots outside of pronounced degradation of mutant CEP135 mRNA the centrosome that were not positive for pericentrin (Figure S5A). In contrast, a smaller PCR product of only (Figure 3A). By contrast, we did not detect the mutant 385 bp, generated with primers just flanking the site of protein on the centrosome; instead, in a few cells we mutation, was easily obtained from both control and observed a diffuse cytoplasmic staining (Figure 3B). Over- mutant RNA (Figure S5B). A contamination of the mutant

The American Journal of Human Genetics 90, 871–878, May 4, 2012 875 Figure 4. Centrosomal and Spindle Abnormalities in COS-7 Cells Transiently Expressing Wild-Type and Mutant CEP135 (A) A GFP-CEP135-expressing cell at metaphase showing bipolar spindles with two centrosomes. (B) GFP-CEP135-mut-expressing cell with supernumerary centrosomes, which result in multipolar spindle formation. Centrosomes were detected by pericentrin-specific antibodies (turquoise). Scale bar, 5 mm. sample with wild-type RNA was excluded by sequencing a reduced growth rate.15 In mutant primary fibroblast cells, of the PCR product. One explanation to reconcile these we have observed strongly reduced growth as well, which apparently contradictory results might be an incomplete did not allow the acquisition of further data. The two decay of the mutant mRNA. To verify this hypothesis, we known interaction partners of CEP135, the 50 kDa subunit performed quantitative real-time PCR with cDNAs from of the dynactin complex (p50) and C-Nap1, have their patient V-1 and his mother (IV-2), along with two different binding sites in the C-terminal domain of CEP135. A trun- control samples. GAPDH and CEP135 gene-specific primers cation of CEP135 releases p50 and C-Nap1 and causes (Table S8), 10 mM each, were mixed with SYBR Green decomposition of functional centrosomes and premature PCR Master Mix (QIAGEN) and the respective cDNA. An centrosome splitting, respectively.23,24 Knockdown of ANXA7 was used for calibration. Thermal cycler CEP135 leads to disorganized interphase and mitotic conditions were 95C for 15 min, 41 cycles of 94C for spindle microtubules with bipolar and multipolar orienta- 30 s, 57C for 45 s, and 68C for 40 s. A DNA Engine tion.15 A role in procentriole formation has been uncov- Opticon 2 (MJ Research, Bio-Rad) was used. The data ered wherein CEP135 and CPAP, also known as CENPJ were analyzed with MJ Opticon Monitor software (version (MCPH6 protein), form a core structure within the prox- 3.1). We found a dramatic reduction in the CEP135 mRNA imal lumen of both parental and nascent centrioles.16 level in the patient as compared to the controls (Figure S6). The phenotype of multiple centrosomes has also been The mother’s CEP135 mRNA level was twice as high as that described in different patient and animal cells carrying of the affected son but still significantly reduced when mutations in genes responsible for MCPH. Approximately compared to the controls. These data corroborate the 25% of the cells of lymphoblastoid cell lines carrying notion of a partial NMD effect caused by the mutation a mutation in MCPH1 harbored supernumerary centro- c.970delC. somes.25 An aberrant centrosome number was also CEP135 was identified as a centrosomal component observed in Mcph1 null DT40 cells after by proteomic analysis and shown to be present in the treatment.26 Mutant centrosomal protein CEP152 is the pericentriolar matrix, around the centriolar surface, and cause of primary microcephaly MCPH4 and of Seckel within the proximal lumen of the centrioles.16,20 In syndrome.6,27 Fibroblast cells of patients with Seckel syn- Chlamydomonas, the CEP135 ortholog Bld10p localizes drome associated with CEP152 mutations also harbored to the cartwheel, a 9-fold symmetrical structure that multiple centrosomes of variable size.27 Cdk5rap2 mutant presumably functions as the scaffold for the centriole- mouse embryonic fibroblasts had supernumerary centro- microtubule assembly and is responsible for achieving somes resulting in bipolar and multipolar spindles.28 the 9-fold symmetry.21 Bld10p (bld10) show aber- A similar phenotype was seen in mouse neuronal progen- rant interphase microtubules and mitotic spindles, defects itors mutated in the MCPH3-associated gene ortholog in cell division, and a significant reduction of the growth Cdk5rap2.29 We have identified multiple centrosomes rate.22 Knockdown of CEP135 in CHO cells also led to in primary CEP135 patient fibroblasts as well as in cells

876 The American Journal of Human Genetics 90, 871–878, May 4, 2012 transfected with a plasmid encoding the mutant protein. Online Mendelian Inheritance in Man (OMIM), http://www. The abnormal centrosome number supports CEP135’s omim.org role in centriole biogenesis, whereas a disorganization of SMART database, http://smart.embl-heidelberg.de/ the microtubule network points to its role at the centro- UCSC Genome Browser, http://genome.ucsc.edu some as microtubule-organizing center. In summary, we have shown a truncating mutation of CEP135 to cause autosomal-recessive primary micro- References cephaly in a Pakistani family. We suggest designation of 1. Kaindl, A.M., Passemard, S., Kumar, P., Kraemer, N., Issa, L., the corresponding locus at 4q12 MCPH8. The correspond- Zwirner, A., Gerard, B., Verloes, A., Mani, S., and Gressens, P. ing protein, CEP135, is a centrosomal component and (2010). Many roads lead to primary autosomal recessive further strengthens the role of centrosomes in the cause microcephaly. Prog. Neurobiol. 90, 363–383. of MCPH. In 18% and 22% of mutant fibroblasts, we 2. Jackson, A.P., Eastwood, H., Bell, S.M., Adu, J., Toomes, C., observed either the presence of multiple centrosomes or Carr, I.M., Roberts, E., Hampshire, D.J., Crow, Y.J., Mighell, a complete loss of centrosomes, respectively. Centrosome A.J., et al. (2002). Identification of , a protein amplification most likely affects mitotic progression and implicated in determining the size of the . Am. J. Hum. Genet. 71, 136–142. mitotic spindle orientation and leads to multipolar spin- 3. Nicholas, A.K., Khurshid, M., De´sir, J., Carvalho, O.P., Cox, J.J., dles, which result in the loss of progenitor cells. This has MCPH1 CEP152 Thornton, G., Kausar, R., Ansar, M., Ahmad, W., Verloes, A., been observed in and mutant human cells et al. (2010). WDR62 is associated with the spindle pole and Cdk5rap2 25–28 as well as in mutant mouse cells. These is mutated in human microcephaly. Nat. Genet. 42, 1010– events can also affect the polarity of cell division in the 1014. neural progenitor cells, leading to an altered number of 4. Yu, T.W., Mochida, G.H., Tischfield, D.J., Sgaier, S.K., Flores- neural progenitor cells or an altered fate, which may be Sarnat, L., Sergi, C.M., Topc¸u, M., McDonald, M.T., Barry, the cause of reduced production. B.J., Felie, J.M., et al. (2010). Mutations in WDR62, encoding a centrosome-associated protein, cause microcephaly with simplified gyri and abnormal cortical architecture. Nat. Genet. Supplemental Data 42, 1015–1020. Supplemental Data include six figures and eight tables and can be 5. Bond, J., Roberts, E., Springell, K., Lizarraga, S.B., Scott, S., found with this article online at http://www.cell.com/AJHG/. Higgins, J., Hampshire, D.J., Morrison, E.E., Leal, G.F., Silva, E.O., et al. (2005). 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