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• W2045767540 abstract "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 genes of the seven known loci code for centrosomal proteins. We studied a family from northern Pakistan with two microcephalic children using homozygosity mapping and found suggestive linkage for regions on chromosomes 2, 4, and 9. We sequenced two positional candidate genes and identified a homozygous frameshift mutation in the gene encoding the 135 kDa centrosomal protein (CEP135), located in the linkage interval on chromosome 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 fragmented centrosomes, disorganized microtubules, 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 mutant protein. Our findings suggest an additional locus 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) 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 genes of the seven known loci code for centrosomal proteins. We studied a family from northern Pakistan with two microcephalic children using homozygosity mapping and found suggestive linkage for regions on chromosomes 2, 4, and 9. We sequenced two positional candidate genes and identified a homozygous frameshift mutation in the gene encoding the 135 kDa centrosomal protein (CEP135), located in the linkage interval on chromosome 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 fragmented centrosomes, disorganized microtubules, 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 mutant protein. Our findings suggest an additional locus 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 251200]) is a neurodevelopmental disorder characterized by reduced size of the cerebral cortex and mild to moderate intellectual disability whereas the architecture of the brain is largely normal. Head circumference is already reduced at birth and usually more than 3 SD below the age- and sex-matched population mean throughout the patient's lifetime. Many patients have a receding forehead. MCPH is heterogeneous; it has seven known loci, MCPH1–MCPH7,1Kaindl A.M. Passemard S. Kumar P. Kraemer N. Issa L. Zwirner A. Gerard B. Verloes A. Mani S. Gressens P. Many roads lead to primary autosomal recessive microcephaly.Prog. Neurobiol. 2010; 90: 363-383Crossref PubMed Scopus (162) Google Scholar each of which is associated with an underlying genetic defect: MCPH1 (MIM 251200) is associated with mutations of MCPH1 (MIM 607117);2Jackson A.P. Eastwood H. Bell S.M. Adu J. Toomes C. Carr I.M. Roberts E. Hampshire D.J. Crow Y.J. Mighell A.J. et al.Identification of microcephalin, a protein implicated in determining the size of the human brain.Am. J. Hum. Genet. 2002; 71: 136-142Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar MCPH2 with or without cortical malformations (MIM 604317) is associated with mutations of WDR62 (MIM 613583);3Nicholas A.K. Khurshid M. Désir J. Carvalho O.P. Cox J.J. Thornton G. Kausar R. Ansar M. Ahmad W. Verloes A. et al.WDR62 is associated with the spindle pole and is mutated in human microcephaly.Nat. Genet. 2010; 42: 1010-1014Crossref PubMed Scopus (226) Google Scholar, 4Yu T.W. Mochida G.H. Tischfield D.J. Sgaier S.K. Flores-Sarnat L. Sergi C.M. Topçu M. McDonald M.T. Barry B.J. Felie J.M. et al.Mutations in WDR62, encoding a centrosome-associated protein, cause microcephaly with simplified gyri and abnormal cortical architecture.Nat. Genet. 2010; 42: 1015-1020Crossref PubMed Scopus (229) Google Scholar MCPH3 (MIM 604804) is associated with mutations of CDK5RAP2 (MIM 608201);5Bond J. Roberts E. Springell K. Lizarraga S.B. Scott S. Higgins J. Hampshire D.J. Morrison E.E. Leal G.F. Silva E.O. et al.A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size.Nat. Genet. 2005; 37: 353-355Crossref PubMed Scopus (438) Google Scholar MCPH4 (MIM 604321) is associated with mutations of CEP152 (MIM 613529);6Guernsey D.L. Jiang H. Hussin J. Arnold M. Bouyakdan K. Perry S. Babineau-Sturk T. Beis J. Dumas N. Evans S.C. et al.Mutations in centrosomal protein CEP152 in primary microcephaly families linked to MCPH4.Am. J. Hum. Genet. 2010; 87: 40-51Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar MCPH5 (MIM 608716) is associated with mutations of ASPM (MIM 605481);7Bond J. Roberts E. Mochida G.H. Hampshire D.J. Scott S. Askham J.M. Springell K. Mahadevan M. Crow Y.J. Markham A.F. et al.ASPM is a major determinant of cerebral cortical size.Nat. Genet. 2002; 32: 316-320Crossref PubMed Scopus (459) Google Scholar MCPH6 (MIM 608393) is associated with mutations of CENPJ (MIM 609279);5Bond J. Roberts E. Springell K. Lizarraga S.B. Scott S. Higgins J. Hampshire D.J. Morrison E.E. Leal G.F. Silva E.O. et al.A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size.Nat. Genet. 2005; 37: 353-355Crossref PubMed Scopus (438) Google Scholar and MCPH7 (MIM 612703) is associated with mutations of STIL (MIM 181590).8Kumar A. Girimaji S.C. Duvvari M.R. Blanton S.H. Mutations in STIL, encoding a pericentriolar and centrosomal protein, cause primary microcephaly.Am. J. Hum. Genet. 2009; 84: 286-290Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar Recently, a new locus at HSA 10q11.23-21.3 was described in a consanguineous Turkish family, but the authors were not able to find the disease-causing gene variant.9Marchal J.A. Ghani M. Schindler D. Gavvovidis I. Winkler T. Esquitino V. Sternberg N. Busche A. Krawitz P. Hecht J. et al.Misregulation of mitotic chromosome segregation in a new type of autosomal recessive primary microcephaly.Cell Cycle. 2011; 10: 2967-2977Crossref PubMed Scopus (11) Google Scholar The MCPH-associated genes described to date have been implicated in cell division and cell cycle regulation, and many of the corresponding gene products are localized to the centrosome. It has been speculated that they affect neural progenitor cell number through disturbed microtubule organization at the centrosome, resulting in altered cell division and cortical development. Despite their similar subcellular localization, the biological functions of these genes may vary.1Kaindl A.M. Passemard S. Kumar P. Kraemer N. Issa L. Zwirner A. Gerard B. Verloes A. Mani S. Gressens P. Many roads lead to primary autosomal recessive microcephaly.Prog. Neurobiol. 2010; 90: 363-383Crossref PubMed Scopus (162) Google Scholar, 3Nicholas A.K. Khurshid M. Désir J. Carvalho O.P. Cox J.J. Thornton G. Kausar R. Ansar M. Ahmad W. Verloes A. et al.WDR62 is associated with the spindle pole and is mutated in human microcephaly.Nat. Genet. 2010; 42: 1010-1014Crossref PubMed Scopus (226) Google Scholar, 4Yu T.W. Mochida G.H. Tischfield D.J. Sgaier S.K. Flores-Sarnat L. Sergi C.M. Topçu M. McDonald M.T. Barry B.J. Felie J.M. et al.Mutations in WDR62, encoding a centrosome-associated protein, cause microcephaly with simplified gyri and abnormal cortical architecture.Nat. Genet. 2010; 42: 1015-1020Crossref PubMed Scopus (229) Google Scholar, 10Thornton G.K. Woods C.G. Primary microcephaly: do all roads lead to Rome?.Trends Genet. 2009; 25: 501-510Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar We studied a consanguineous family from northern Pakistan; the two affected children had primary microcephaly at birth (Figures 1A and 1B). After informed consent of the parents was obtained, pedigree information was documented and clinical data and blood samples were collected from the affected siblings, their father, and an unaffected sibling. Each affected child had a sloping forehead. By 5 years of age they showed severe cognitive deficits; their speech was not understandable. They were unable to form sentences and even to say any clear words, although their hearing was not impaired. Other abnormalities were not apparent. Individual V-2 died at 11 years. The head circumference of the affected individuals ranged between −12 and −14.5 SD compared to the average population of the same age and sex. The parents were healthy with normal head circumference. Ethical approval for this study was obtained from the ethics review board at the National Institute for Biotechnology and Genetic Engineering in Faisalabad according to the Declaration of Helsinki. Standard procedures were used to extract DNA from the blood samples for homozygosity mapping. Initially, we excluded all known MCPH loci in the family, using STR markers to demonstrate heterozygosity in the relevant genomic regions. We then performed a genome-wide linkage analysis using the Affymetrix GeneChip Human Mapping 250K Sty Array. Data handling, evaluation, and statistical analysis were performed as described previously.11Borck G. Ur Rehman A. Lee K. Pogoda H.M. Kakar N. von Ameln S. Grillet N. Hildebrand M.S. Ahmed Z.M. Nürnberg G. et al.Loss-of-function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42.Am. J. Hum. Genet. 2011; 88: 127-137Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar We observed three peaks on chromosomes 2, 4, and 9 that were suggestive for linkage with maximum multipoint LOD scores of 2.07, 2.53, and 2.53, respectively (Figure S1). The underlying common homozygous regions defined a candidate region of 8.6 Mb on chromosome 2 (90,183,484–98,795,792; hg19), a candidate region of 18.1 Mb on chromosome 4 (40,631,476–58,702,130; hg19), and a candidate region of 8.8 Mb on chromosome 9 (92,274,161–101,122,314; hg19). Altogether these regions include more than 200 annotated known and predicted coding genes (UCSC Genome Bioinformatics, hg19). The genes were prioritized with Endeavour and GeneWanderer.12Aerts S. Lambrechts D. Maity S. Van Loo P. Coessens B. De Smet F. Tranchevent L.C. De Moor B. Marynen P. Hassan B. et al.Gene prioritization through genomic data fusion.Nat. Biotechnol. 2006; 24: 537-544Crossref PubMed Scopus (702) Google Scholar, 13Köhler S. Bauer S. Horn D. Robinson P.N. Walking the interactome for prioritization of candidate disease genes.Am. J. Hum. Genet. 2008; 82: 949-958Abstract Full Text Full Text PDF PubMed Scopus (928) Google Scholar Highly ranked genes were further scrutinized manually with the NCBI, Ensembl, and UCSC genome databases. Finally, we gave top priority to genes that were very likely to have important functions related to cell division or chromosome segregation and decided to sequence the following two strong candidate genes first: the cell division cycle-14 homolog B gene (CDC14B [MIM 603505]) at cytoband 9q22.32-q31.1 and the gene encoding the 135 kDa centrosomal protein (CEP135 [MIM 611423]) at HAS 4p14-q12 (Figure 1C). All exons and the intron-exon boundaries of CEP135 and CDC14B were sequenced in the two affected individuals. The PCR products (primers are listed in Table S1) were sequenced bidirectionally with a BigDye Terminator v1.1 cycle sequencing kit on an ABI3730xl automated DNA sequencer. Sequences were analyzed with DNASTAR (Lasergene) and Mutation Surveyor (SoftGenetics). We found no mutation in CDC14B but a homozygous single base-pair (bp) deletion (c.970delC) in exon 8 of CEP135. We found the homozygous 1 bp deletion in both affected children, but not in the unaffected child, whereas the father was heterozygous for this mutation (Figure 1D), which is compatible with recessive inheritance. Moreover, the mutation is unlikely to be a polymorphism, as it is not listed in dbSNP, and we could not find it when testing 384 healthy Pakistani controls with pyrosequencing. For this purpose, PCR primers (listed in Table S2) were designed by the PSQ Assay Design program v.1.0.6 (QIAGEN, Hilden, Germany). Pyrosequencing was done according to the manufacturer's instructions on a PSQ HS96A instrument (QIAGEN) with the use of PyroMark Gold Q96 Reagents (QIAGEN). The data were analyzed by Pyro Q-CpG v.1.0.9 analysis software (QIAGEN). In an attempt to identify a second independent mutation of CEP135, we sequenced CEP135 in patients from seven other families affected with MCPH from northern Pakistan in which all known MCPH-associated genes were previously excluded. No CEP135 mutation was found in any of these families. Therefore, we decided to perform whole-exome sequencing of the affected boy (individual V-1) of the family presented in Figure 1B in order to demonstrate that there were no other mutations in relevant genes that might also explain the phenotype. We fragmented 1 μg of DNA using sonification technology (Covaris, Woburn, MA, USA). The fragments were end repaired and adaptor ligated. After size selection, the library was subjected to the enrichment process. We chose the SeqCap EZ Human Exome Library v2.0 kit from NimbleGen (Roche NimbleGen, Madison, WI, USA) and analyzed the sample on an Illumina HiSeq 2000 sequencing instrument. About 10 Gb of sequence were produced for this sample by loading it individually on one lane of a flow cell and generating paired-end reads of 2 × 100 bp. This resulted in a very high coverage; i.e., > 30× for nearly 92% of the target sequences, which comprised about 44 Mb. Primary data were filtered according to signal purity by the Illumina Realtime Analysis (RTA) software v1.8. Subsequently, the reads were mapped to the human genome reference build hg19 via the ELANDv2 alignment algorithm on a multinode compute cluster. With the use of CASAVA v1.8, PCR duplicates were filtered out, and the output was converted into BAM format. Variant calling was performed with the use of SAMtools (version 0.1.7) for indel detection.14Li H. Handsaker B. Wysoker A. Fennell T. Ruan J. Homer N. Marth G. Abecasis G. Durbin R. 1000 Genome Project Data Processing SubgroupThe Sequence Alignment/Map format and SAMtools.Bioinformatics. 2009; 25: 2078-2079Crossref PubMed Scopus (31547) Google Scholar Scripts developed in-house at the Cologne Center for Genomics were applied to detect protein changes, affected splice sites, and overlaps with known variants. In particular, we filtered the variants for high-quality unknown variants in the linkage intervals (dbSNP build 132 or the 1000 Genomes database; in-house variation database; and public Exome Variant Server, NHLBI Exome Sequencing Project, Seattle) (Table S3). Only three other homozygous variants resisted our filter criteria in addition to CEP135 c.970delC. None of these could be assumed to be relevant for the phenotype (Table S4). Likewise, we could not find deleterious mutations in any of the seven known MCPH-associated genes, even when allowing for compound heterozygosity during filtering. The deletion c.970delC of CEP135 results in a frameshift, changing glutamine at position 324 into serine, immediately followed by a premature termination codon (p.Gln324Serfs∗2). This truncating mutation is obviously incompatible with a normal function of CEP135 if one considers the size of the protein and the position of the mutation (Figures 1E and 1F). CEP135 consists of 26 exons, and its open reading frame codes for a polypeptide of 1,140 amino acids. CEP135 is a conserved α-helical protein which is present at the centrosome throughout the cell cycle. Electron-microscopic studies showed its association with the pericentriolar material, an electron-dense material surrounding the centrioles. Reducing CEP135 amounts in cells via RNA interference caused a disorganization of interphase and mitotic spindles, leading to the hypothesis that CEP135 has a role in maintaining the structure and organization of the centrosome and of microtubules.15Ohta T. Essner R. Ryu J.H. Palazzo R.E. Uetake Y. Kuriyama R. Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.J. Cell Biol. 2002; 156: 87-99Crossref PubMed Scopus (121) Google Scholar More recently the protein was identified as a centriolar component; it functions in centriole biogenesis and presumably has a scaffolding role.16Kleylein-Sohn J. Westendorf J. Le Clech M. Habedanck R. Stierhof Y.D. Nigg E.A. Plk4-induced centriole biogenesis in human cells.Dev. Cell. 2007; 13: 190-202Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar Centrioles are core components of animal centrosomes and act as basal bodies to assemble cilia and flagella.17Azimzadeh J. Marshall W.F. Building the centriole.Curr. Biol. 2010; 20: R816-R825Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar To unravel the effect of the mutation on the cellular level, we analyzed control and patient fibroblasts. Biopsies were taken from the affected individual V-1 (Figure 1B) and healthy individuals. Tissues were cleaned with antiseptic agent (Betaisodona, Mundipharma) and incubated overnight with Dispase II (1.5 U/ml, Roche) diluted in PBS, pH 6.8, at 4°C to separate the intact epidermis from the dermis. The dermis was incubated in Dulbecco's modified Eagle's medium (DMEM) at 37°C. After one week, fibroblasts were detected. The pool of fibroblasts was increased by additional culturing. Primary fibroblasts established from the patient grew very slowly. To enhance growth, DMEM with 15% fetal bovine serum was used. Patient and wild-type primary fibroblasts were then cultured on 12 mm coverslips and fixed with 3% paraformaldehyde. For staining of microtubules, cells were incubated for 15 min in tubulin stabilization buffer, which is composed of Hank's buffer (137 mM NaCl, 5 mM KCl, 1.1 mM Na2HPO4, 0.4 mM KH2PO4, 5 mM Glucose, 4 mM NaHCO3) containing 1 mM MES, pH 6.8, 2 mM EGTA, and 2 mM MgCl2. Permeabilization was done by 0.5% Triton X-100 in 1× tubulin stabilization buffer for 4 min at room temperature. For γ-tubulin detection, the cells were fixed with prechilled methanol for 10 min at −20°C. Subsequently, the fixed cells were treated three times with tubulin stabilization buffer. Blocking was done for 15 min with blocking buffer (1× PBG: PBS containing 5% BSA and 0.45% fish gelatin). Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. The following antibodies were used: mouse monoclonal anti γ-tubulin (Sigma-Aldrich, GTU-88; 1:300), rabbit polyclonal anti-pericentrin (Abcam, ab4448; 1:300), and rat monoclonal anti α-tubulin (YL 1/2 1:20).18Kilmartin J.V. Wright B. Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line.J. Cell Biol. 1982; 93: 576-582Crossref PubMed Scopus (636) Google Scholar After incubation, samples were treated with 1× PBS three times for 5 min followed by secondary antibody incubation (1:1,000 diluted in blocking buffer) for one hour at room temperature. Alexa Fluor 568 goat anti-mouse IgG (Invitrogen, A11004), Alexa Fluor 647 donkey anti-mouse IgG (Invitrogen, A31571), and Alexa Fluor 488 goat anti-rat IgG (Invitrogen, A11006) were used as secondary antibodies. DNA was detected with DAPI (Sigma-Aldrich, D9564). Finally, the cells were mounted on glass slides with Gelvatol. Images were taken with a confocal microscope (Leica, LSM TCS SP5). Control primary fibroblasts had oval nuclei surrounded by organized microtubules and a single centrosome in 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 appeared fragmented (Figure S2). The microtubule network was frequently disorganized (∼55% of the cells), which was accompanied by cell shape changes (Figures 2C and S3; Table S5). We also observed misshapen and fragmented nuclei (Figure 2E and S4). Statistical analysis showed that ∼20% of the mutant cells harbored misshapen nuclei as compared to ∼3% in control fibroblasts (Figure 2F; Table S5). Another prominent aspect of the patient's primary fibroblasts was the complete loss of centrosomes. Approximately 22% of mutant primary fibroblasts were without centrosomes as detected with γ-tubulin, whereas this was never observed in control cells (Table S5; Figure S3). Most cells with misshapen nuclei were also devoid of centrosomes (Figure S4). For ectopic expression of wild-type and mutant (c.970delC) CEP135 fused to green fluorescent protein (GFP), we used COS-7 cells. Gateway Technology (Invitrogen) was employed to clone wild-type (NM_025009.3) CEP135 cDNA. The CEP135 mutation (c.970delC) was introduced into the full-length cDNA with the use of the QuikChange II Site-Directed Mutagenesis Kit (Stratagene) (primers are listed in Table S6) in an attempt to reproduce the patient's situation. Entry clones in pENTR/TEV/D-TOPO were transformed into Gateway destination vector pcDNA-DEST53, which has an N-terminal cycle-3 GFP tag. Both wild-type and mutant plasmids (10 μg/μl) were used for transfection of COS-7 cells. The Gene Pulser II (Bio-Rad) device was used for electroporation. 72 hr after transfection, the cells were subjected to immunofluorescence. When we analyzed the localization of the proteins and their effect on centrosomes and microtubule organization, we observed wild-type GFP-tagged CEP135 at the centrosome where it colocalized with the centrosomal protein pericentrin. We also detected it in some spots outside of the centrosome that were not positive for pericentrin (Figure 3A ). By contrast, we did not detect the mutant protein on the centrosome; instead, in a few cells we observed a diffuse cytoplasmic staining (Figure 3B). Overexpression 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 multiple centrosomes (up to 5), which then resulted in multipolar spindle formation. This phenotype was not observed in the cells that overexpressed the wild-type protein. Interestingly, the GFP-tagged CEP135 was detected on microtubules (Figure 4). These findings of a disorganized microtubule network and multiple centrosomes in cells transfected with GFP-tagged CEP135 resembled those observed in mutant primary fibroblast cells.Figure 4Centrosomal and Spindle Abnormalities in COS-7 Cells Transiently Expressing Wild-Type and Mutant CEP135Show full caption(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 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (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 μm. The CEP135 mutation c.970delC is thought to lead to a truncation of the protein due to the introduction of a premature stop codon (p.Gln324Serfs∗2). Alternatively, it might also trigger nonsense-mediated mRNA decay (NMD).19Nicholson P. Mühlemann O. Cutting the nonsense: the degradation of PTC-containing mRNAs.Biochem. Soc. Trans. 2010; 38: 1615-1620Crossref PubMed Scopus (88) Google Scholar We designed RT-PCR experiments to investigate this (Table S7). The PAXgene Blood RNA system (QIAGEN) was used to extract RNA from patient and control blood samples, and cDNA was synthesized with SuperScript III reverse transcriptase enzyme (Invitrogen). When primers were used to amplify a nearly full-length CEP135 cDNA of ∼3.4 kb, no PCR product was obtained with the patient's RNA, but only with the control sample, suggesting pronounced degradation of mutant CEP135 mRNA (Figure S5A). In contrast, a smaller PCR product of only 385 bp, generated with primers just flanking the site of mutation, was easily obtained from both control and mutant RNA (Figure S5B). A contamination of the mutant sample with wild-type RNA was excluded by sequencing of the PCR product. One explanation to reconcile these apparently contradictory results might be an incomplete decay of the mutant mRNA. To verify this hypothesis, we performed quantitative real-time PCR with cDNAs from patient V-1 and his mother (IV-2), along with two different control samples. GAPDH and CEP135 gene-specific primers (Table S8), 10 μM each, were mixed with SYBR Green PCR Master Mix (QIAGEN) and the respective cDNA. An ANXA7 plasmid was used for calibration. Thermal cycler conditions were 95°C for 15 min, 41 cycles of 94°C for 30 s, 57°C for 45 s, and 68°C for 40 s. A DNA Engine Opticon 2 (MJ Research, Bio-Rad) was used. The data were analyzed with MJ Opticon Monitor software (version 3.1). We found a dramatic reduction in the CEP135 mRNA level in the patient as compared to the controls (Figure S6). The mother's CEP135 mRNA level was twice as high as that of the affected son but still significantly reduced when compared to the controls. These data corroborate the notion of a partial NMD effect caused by the mutation c.970delC. CEP135 was identified as a centrosomal component by proteomic analysis and shown to be present in the pericentriolar matrix, around the centriolar surface, and within the proximal lumen of the centrioles.16Kleylein-Sohn J. Westendorf J. Le Clech M. Habedanck R. Stierhof Y.D. Nigg E.A. Plk4-induced centriole biogenesis in human cells.Dev. Cell. 2007; 13: 190-202Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar, 20Andersen J.S. Wilkinson C.J. Mayor T. Mortensen P. Nigg E.A. Mann M. Proteomic characterization of the human centrosome by protein correlation profiling.Nature. 2003; 426: 570-574Crossref PubMed Scopus (1051) Google Scholar In Chlamydomonas, the CEP135 ortholog Bld10p localizes to the cartwheel, a 9-fold symmetrical structure that presumably functions as the scaffold for the centriole-microtubule assembly and is responsible for achieving the 9-fold symmetry.21Hiraki M. Nakazawa Y. Kamiya R. Hirono M. Bld10p constitutes the cartwheel-spoke tip and stabilizes the 9-fold symmetry of the centriole.Curr. Biol. 2007; 17: 1778-1783Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar Bld10p mutants (bld10) show aberrant interphase microtubules and mitotic spindles, defects in cell division, and a significant reduction of the growth rate.22Matsuura K. Lefebvre P.A. Kamiya R. Hirono M. Bld10p, a novel protein essential for basal body assembly in Chlamydomonas: localization to the cartwheel, the first ninefold symmetrical structure appearing during assembly.J. Cell Biol. 2004; 165: 663-671Crossref PubMed Scopus (109) Google Scholar Knockdown of CEP135 in CHO cells also led to a reduced growth rate.15Ohta T. Essner R. Ryu J.H. Palazzo R.E. Uetake Y. Kuriyama R. Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.J. Cell Biol. 2002; 156: 87-99Crossref PubMed Scopus (121) Google Scholar In mutant primary fibroblast cells, we have observed strongly reduced growth as well, which did not allow the acquisition of further data. The two known interaction partners of CEP135, the 50 kDa subunit of the dynactin complex (p50) and C-Nap1, have their binding sites in the C-terminal domain of CEP135. A truncation of CEP135 releases p50 and C-Nap1 and causes decomposition of functional centrosomes and premature centrosome splitting, respectively.23Uetake Y. Terada Y. Matuliene J. Kuriyama R. Interaction of Cep135 with a p50 dynactin subunit in mammalian centrosomes.Cell Motil. Cytoskeleton. 2004; 58: 53-66Crossref PubMed Scopus (28) Google Scholar, 24Kim K. Lee S. Chang J. Rhee K. A novel function of CEP135 as a platform protein of C-NAP1 for its centriolar localization.Exp. Cell Res. 2008; 314: 3692-3700Crossref PubMed Scopus (48) Google Scholar Knockdown of CEP135 leads to disorganized interphase and mitotic spindle microtubules with bipolar and multipolar orientation.15Ohta T. Essner R. Ryu J.H. Palazzo R.E. Uetake Y. Kuriyama R. Characterization of Cep135, a novel coiled-coil centrosomal protein involved in microtubule organization in mammalian cells.J. Cell Biol. 2002; 156: 87-99Crossref PubMed Scopus (121) Google Scholar A role in procentriole formation has been uncovered wherein CEP135 and CPAP, also known as CENPJ (MCPH6 protein), form a core structure within the proximal lumen of both parental and nascent centrioles.16Kleylein-Sohn J. Westendorf J. Le Clech M. Habedanck R. Stierhof Y.D. Nigg E.A. Plk4-induced centriole biogenesis in human cells.Dev. Cell. 2007; 13: 190-202Abstract Full Text Full Text PDF PubMed Scopus (497) Google Scholar The phenotype of multiple centrosomes has also been described in different patient and animal cells carrying mutations in genes responsible for MCPH. Approximately 25% of the cells of lymphoblastoid cell lines carrying a mutation in MCPH1 harbored supernumerary centrosomes.25Alderton G.K. Galbiati L. Griffith E. Surinya K.H. Neitzel H. Jackson A.P. Jeggo P.A. O'Driscoll M. Regulation of mitotic entry by microcephalin and its overlap with ATR signalling.Nat. Cell Biol. 2006; 8: 725-733Crossref PubMed Scopus (146) Google Scholar An aberrant centrosome number was also observed in Mcph1 null DT40 cells after ionizing radiation treatment.26Brown J.A.L. Bourke E. Liptrot C. Dockery P. Morrison C.G. MCPH1/BRIT1 limits ionizing radiation-induced centrosome amplification.Oncogene. 2010; 29: 5537-5544Crossref PubMed Scopus (27) Google Scholar Mutant centrosomal protein CEP152 is the cause of primary microcephaly MCPH4 and of Seckel syndrome.6Guernsey D.L. Jiang H. Hussin J. Arnold M. Bouyakdan K. Perry S. Babineau-Sturk T. Beis J. Dumas N. Evans S.C. et al.Mutations in centrosomal protein CEP152 in primary microcephaly families linked to MCPH4.Am. J. Hum. Genet. 2010; 87: 40-51Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 27Kalay E. Yigit G. Aslan Y. Brown K.E. Pohl E. Bicknell L.S. Kayserili H. Li Y. Tüysüz B. Nürnberg G. et al.CEP152 is a genome maintenance protein disrupted in Seckel syndrome.Nat. Genet. 2011; 43: 23-26Crossref PubMed Scopus (177) Google Scholar Fibroblast cells of patients with Seckel syndrome associated with CEP152 mutations also harbored multiple centrosomes of variable size.27Kalay E. Yigit G. Aslan Y. Brown K.E. Pohl E. Bicknell L.S. Kayserili H. Li Y. Tüysüz B. Nürnberg G. et al.CEP152 is a genome maintenance protein disrupted in Seckel syndrome.Nat. Genet. 2011; 43: 23-26Crossref PubMed Scopus (177) Google Scholar Cdk5rap2 mutant mouse embryonic fibroblasts had supernumerary centrosomes resulting in bipolar and multipolar spindles.28Barrera J.A. Kao L.R. Hammer R.E. Seemann J. Fuchs J.L. Megraw T.L. CDK5RAP2 regulates centriole engagement and cohesion in mice.Dev. Cell. 2010; 18: 913-926Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar A similar phenotype was seen in mouse neuronal progenitors mutated in the MCPH3-associated gene ortholog Cdk5rap2.29Lizarraga S.B. Margossian S.P. Harris M.H. Campagna D.R. Han A.P. Blevins S. Mudbhary R. Barker J.E. Walsh C.A. Fleming M.D. Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors.Development. 2010; 137: 1907-1917Crossref PubMed Scopus (201) Google Scholar We have identified multiple centrosomes in primary CEP135 patient fibroblasts as well as in cells transfected with a plasmid encoding the mutant protein. The abnormal centrosome number supports CEP135's role in centriole biogenesis, whereas a disorganization of the microtubule network points to its role at the centrosome as microtubule-organizing center. In summary, we have shown a truncating mutation of CEP135 to cause autosomal-recessive primary microcephaly in a Pakistani family. We suggest designation of the corresponding locus at 4q12 MCPH8. The corresponding protein, CEP135, is a centrosomal component and further strengthens the role of centrosomes in the cause of MCPH. In 18% and 22% of mutant fibroblasts, we observed either the presence of multiple centrosomes or a complete loss of centrosomes, respectively. Centrosome amplification most likely affects mitotic progression and mitotic spindle orientation and leads to multipolar spindles, which result in the loss of progenitor cells. This has been observed in MCPH1 and CEP152 mutant human cells as well as in Cdk5rap2 mutant mouse cells.25Alderton G.K. Galbiati L. Griffith E. Surinya K.H. Neitzel H. Jackson A.P. Jeggo P.A. O'Driscoll M. Regulation of mitotic entry by microcephalin and its overlap with ATR signalling.Nat. Cell Biol. 2006; 8: 725-733Crossref PubMed Scopus (146) Google Scholar, 26Brown J.A.L. Bourke E. Liptrot C. Dockery P. Morrison C.G. MCPH1/BRIT1 limits ionizing radiation-induced centrosome amplification.Oncogene. 2010; 29: 5537-5544Crossref PubMed Scopus (27) Google Scholar, 27Kalay E. Yigit G. Aslan Y. Brown K.E. Pohl E. Bicknell L.S. Kayserili H. Li Y. Tüysüz B. Nürnberg G. et al.CEP152 is a genome maintenance protein disrupted in Seckel syndrome.Nat. Genet. 2011; 43: 23-26Crossref PubMed Scopus (177) Google Scholar, 28Barrera J.A. Kao L.R. Hammer R.E. Seemann J. Fuchs J.L. Megraw T.L. CDK5RAP2 regulates centriole engagement and cohesion in mice.Dev. Cell. 2010; 18: 913-926Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar These events can also affect the polarity of cell division in the neural progenitor cells, leading to an altered number of neural progenitor cells or an altered fate, which may be the cause of reduced neuron production. We are grateful to all family members for their participation in this study. We wish to thank Ursula Euteneuer and Ludwig Eichinger for helpful discussion and Ingelore Bäßmann, Martina Munck, and Alexandra Herzog for technical assistance. P.N. is a founder, CEO, and shareholder of ATLAS Biolabs GmbH. ATLAS Biolabs GmbH is a service provider for genomic analyses. This work was supported by grants from the Higher Education Commission (HEC) of Pakistan, the German Academic Exchange Service (DAAD), and the Center for Molecular Medicine Cologne (CMMC). Download .pdf (.69 MB) Help with pdf files Document S1. Figures S1–S6 and Tables S1–S8 The URLs for the data presented herein are as follows:Endeavour, http://homes.esat.kuleuven.be/∼bioiuser/endeavour/tool/endeavourweb.phpEnsembl Genome Browser, http://www.ensembl.orgExome Variant Server, http://snp.gs.washington.edu/EVS/GeneWanderer, http://compbio.charite.de/genewanderer/GeneWandererMarshfield genetic map, http://www.bli.uzh.ch/BLI/Projects/genetics/maps/marsh.htmlNCBI Map Viewer, http://www.ncbi.nlm.nih.gov/mapview/Online Mendelian Inheritance in Man (OMIM), http://www.omim.orgSMART database, http://smart.embl-heidelberg.de/UCSC Genome Browser, http://genome.ucsc.edu" @default.
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