REPORT

A Truncating Mutation of CEP135 Causes PrimaryMicrocephaly and Disturbed Centrosomal Function

Muhammad Sajid Hussain,1,2,3,4 Shahid Mahmood Baig,4 Sascha Neumann,2 Gudrun Nurnberg,1,3

Muhammad Farooq,4 Ilyas Ahmad,1,4 Thomas Alef,1 Hans Christian Hennies,1,5 Martin Technau,2

Janine Altmuller,1 Peter Frommolt,1,5 Holger Thiele,1 Angelika Anna Noegel,1,2,3,5,*and Peter Nurnberg1,3,5,*

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 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 centro-

somal 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 showedmultiple and frag-

mented 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 addi-

tional 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 andmild 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 life-

time. Many patients have a receding forehead. MCPH is

heterogeneous; it has seven known loci, MCPH1–

MCPH7,1 each of which is associated with an underlying

genetic defect: MCPH1 (MIM 251200) is associated with

mutations of MCPH1 (MIM 607117);2 MCPH2 with or

without cortical malformations (MIM 604317) is associ-

ated with mutations of WDR62 (MIM 613583);3,4

MCPH3 (MIM 604804) is associated with mutations of

CDK5RAP2 (MIM 608201);5 MCPH4 (MIM 604321) is asso-

ciated with mutations of CEP152 (MIM 613529);6 MCPH5

(MIM 608716) is associated with mutations of ASPM (MIM

605481);7 MCPH6 (MIM 608393) is associated with muta-

tions ofCENPJ (MIM 609279);5 andMCPH7 (MIM 612703)

is associated with mutations of STIL (MIM 181590).8

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.9 The

MCPH-associated genes described to date have been impli-

cated in cell division and cell cycle regulation, andmany of

the corresponding gene products are localized to the

centrosome. It has been speculated that they affect neural

progenitor cell number through disturbed microtubule

1Cologne Center for Genomics (CCG), University of Cologne, 50931 Colog

Cologne, 50931 Cologne, Germany; 3Center for Molecular Medicine Cologne (

nology Division, National Institute for Biotechnology and Genetic Engineer

Cellular Stress Responses in Aging-Associated Diseases (CECAD), University o

*Correspondence: noegel@uni-koeln.de (A.A.N.), nuernberg@uni-koeln.de (P.N

DOI 10.1016/j.ajhg.2012.03.016. �2012 by The American Society of Human

The Am

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.1,3,4,10

We studied a consanguineous family from northern

Pakistan; the two affected children had primary micro-

cephaly 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 fore-

head. By 5 years of age they showed severe cognitive defi-

cits; 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 abnormal-

ities 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 pop-

ulation 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 Engi-

neering in Faisalabad according to the Declaration of Hel-

sinki.

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

ne, Germany; 2Institute of Biochemistry I, Medical Faculty, University of

CMMC), University of Cologne, 50931 Cologne, Germany; 4Health Biotech-

ing (NIBGE), Faisalabad 38000, Pakistan; 5Cologne Excellence Cluster on

f Cologne, 50674 Cologne, Germany

.)

Genetics. All rights reserved.

erican Journal of Human Genetics 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. Onlythe core family is shown. DNA was available for individuals IV-1, V-1, V-2, and V-3.(C) G-banded chromosome 4 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 hisfather (IV-1) show the mutation c.970delC in homozygous and heterozygous status, respectively. The mutant sequences are shownalong with a wild-type trace from a control individual.(E) Schematic representation of the genomic structure of humanCEP135. The 26 exons ofCEP135 are drawn to scale, while introns showjust 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 fourthcoiled-coil domain is indicated.

linkage analysis using the Affymetrix GeneChip Human

Mapping 250K Sty Array. Data handling, evaluation, and

statistical analysis were performed as described previ-

ously.11 We observed three peaks on chromosomes 2, 4,

and 9 that were suggestive for linkage withmaximummul-

tipoint 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,

872 The American Journal of Human Genetics 90, 871–878, May 4, 2

hg19). The genes were prioritized with Endeavour and

GeneWanderer.12,13 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 individ-

uals. The PCR products (primers are listed in Table S1)

012

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 instru-

ment (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 muta-

tion 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 CEP135mutation 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 rele-

vant genes that might also explain the phenotype. We

fragmented 1 mg of DNA using sonification technology

(Covaris, Woburn, MA, USA). The fragments were end re-

paired and adaptor ligated. After size selection, the library

was subjected to the enrichment process.We chose the Seq-

Cap 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 gener-

ating paired-end reads of 23 100 bp. This resulted in a very

high coverage; i.e., > 303 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 multi-

node 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.14

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 partic-

ular, 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

The Am

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, immedi-

ately 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 a-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 sur-

rounding 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.15

More recently the protein was identified as a centriolar

component; it functions in centriole biogenesis and

presumably has a scaffolding role.16 Centrioles are core

components of animal centrosomes and act as basal bodies

to assemble cilia and flagella.17

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 over-

night 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, fibro-

blasts 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 13 tubulin stabilization buffer for 4 min

at room temperature. For g-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 (13 PBG: PBS containing 5% BSA and

erican Journal of Human Genetics 90, 871–878, May 4, 2012 873

Figure 2. Centrosome, Microtubule, andNuclear Shape Defects in CEP135Microce-phalic Patient Cells(A) Immunofluorescence staining andconfocal microscopy images of wild-typeprimary fibroblasts with well-organizedmicrotubules and a single centrosome de-tected by g-tubulin. g-tubulin (turquoise)and a-tubulin (green) antibodies wereused. DAPI (blue) was used for DNA stain-ing. Scale bar, 5 mm.(B) Abnormality of centrosome numberin CEP135 patient primary fibroblasts.Interphase cell showing supernumerarycentrosomes. In this figure three centro-somes were observed as detected byg-tubulin (turquoise). Scale bar, 5 mm.(C) Disorganized microtubule network inpatient fibroblasts. Scale bar, 20 mm.(D) Control primary fibroblast showinga well-shaped ellipsoid nucleus. Scale bar,5 mm.(E) Mutant fibroblast with a dysmorphicnucleus. Scale bar, 10 mm.(F) Graphical representation showingthe number of CEP135 mutant primaryfibroblasts with dysmorphic nuclei. About20% of mutant fibroblasts harboredmisshapen nuclei whereas in control fibro-blasts this number was only ~3%. Threehundred cells of each, wild-type andmutant, were counted. Error bars representSEM, p ¼ 3.44 3 10�03 (Student’s t test).

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

g-tubulin (Sigma-Aldrich, GTU-88; 1:300), rabbit poly-

clonal anti-pericentrin (Abcam, ab4448; 1:300), and rat

monoclonal anti a-tubulin (YL 1/2 1:20).18 After incuba-

tion, samples were treated with 13 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 (Invitro-

gen, A11004), Alexa Fluor 647 donkey anti-mouse IgG

(Invitrogen, A31571), and Alexa Fluor 488 goat anti-rat

IgG (Invitrogen, A11006) were used as secondary anti-

bodies. DNA was detected with DAPI (Sigma-Aldrich,

D9564). Finally, the cells were mounted on glass slides

with Gelvatol. Images were taken with a confocal micro-

scope (Leica, LSM TCS SP5).

Control primary fibroblasts had oval nuclei surrounded

by organized microtubules and a single centrosome in

874 The American Journal of Human Genetics 90, 871–878, May 4, 2012

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). Themicrotubule 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. Approx-

imately 22% of mutant primary fibroblasts were without

centrosomes as detected with g-tubulin, whereas this was

never observed in control cells (Table S5; Figure S3). Most

cells with misshapen nuclei were also devoid of centro-

somes (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 (Invitro-

gen) was employed to clone wild-type (NM_025009.3)

CEP135 cDNA. The CEP135 mutation (c.970delC) was

Figure 3. Distribution of GFP-TaggedWild-Type and Mutant CEP135(A) N-terminally GFP-tagged humanCEP135 (GFP-CEP135) was transientlyexpressed in COS-7 cells. GFP-CEP135is present in dots of variable size andnumber. A single centrosome was detectedby pericentrin-specific antibodies (tur-quoise). Scale bar, 5 mm.(B) COS-7 cell transfected with GFP-taggedmutant CEP135 (GFP-CEP135-mut), wherediffuse staining was detected. Scale bar,5 mm.(C) Abnormal microtubule network inCOS-7 expressing wild-type CEP135. Scalebar, 5 mm.(D) COS-7 cells expressing GFP-CEP135-mut have a disorganized microtubulenetwork. Microtubules appeared brokenand concentrated near the nucleus. Scalebar, 5 mm.

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 mg/ml)

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 immuno-

fluorescence.

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 centro-

some 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). Over-

The American Journal of Huma

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

microtubule network and multiple centrosomes in cells

transfected with GFP-tagged CEP135 resembled those

observed in mutant primary fibroblast cells.

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).19 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

n 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. Centrosomeswere detected by pericentrin-specific antibodies (turquoise). Scale bar, 5 mm.

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 andCEP135 gene-specific primers

(Table S8), 10 mM 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 CEP135mRNA 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.16,20 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.21 Bld10p mutants (bld10) show aber-

rant interphase microtubules and mitotic spindles, defects

in cell division, and a significant reduction of the growth

rate.22 Knockdown of CEP135 in CHO cells also led to

876 The American Journal of Human Genetics 90, 871–878, May 4, 2

a reduced growth rate.15 Inmutant 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 trun-

cation of CEP135 releases p50 and C-Nap1 and causes

decomposition of functional centrosomes and premature

centrosome splitting, respectively.23,24 Knockdown of

CEP135 leads to disorganized interphase and mitotic

spindle microtubules with bipolar and multipolar orienta-

tion.15 A role in procentriole formation has been uncov-

ered wherein CEP135 and CPAP, also known as CENPJ

(MCPH6 protein), form a core structure within the prox-

imal lumen of both parental and nascent centrioles.16

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 centro-

somes.25 An aberrant centrosome number was also

observed in Mcph1 null DT40 cells after ionizing radiation

treatment.26 Mutant centrosomal protein CEP152 is the

cause of primary microcephaly MCPH4 and of Seckel

syndrome.6,27 Fibroblast cells of patients with Seckel syn-

drome associated with CEP152 mutations also harbored

multiple centrosomes of variable size.27 Cdk5rap2 mutant

mouse embryonic fibroblasts had supernumerary centro-

somes resulting in bipolar and multipolar spindles.28

A similar phenotype was seen in mouse neuronal progen-

itors mutated in the MCPH3-associated gene ortholog

Cdk5rap2.29 We have identified multiple centrosomes

in primary CEP135 patient fibroblasts as well as in cells

012

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 centro-

some as microtubule-organizing center.

In summary, we have shown a truncating mutation

of CEP135 to cause autosomal-recessive primary micro-

cephaly in a Pakistani family. We suggest designation of

the corresponding locus at 4q12 MCPH8. The correspond-

ing 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 spin-

dles, which result in the loss of progenitor cells. This has

been observed inMCPH1 and CEP152mutant human cells

as well as in Cdk5rap2 mutant mouse cells.25–28 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.

Supplemental Data

Supplemental Data include six figures and eight tables and can be

found with this article online at http://www.cell.com/AJHG/.

Acknowledgments

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 Baßmann, 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).

Received: November 17, 2011

Revised: March 7, 2012

Accepted: March 15, 2012

Published online: April 19, 2012

Web Resources

The URLs for the data presented herein are as follows:

Endeavour, http://homes.esat.kuleuven.be/~bioiuser/endeavour/

tool/endeavourweb.php

Ensembl Genome Browser, http://www.ensembl.org

Exome Variant Server, http://snp.gs.washington.edu/EVS/

GeneWanderer, http://compbio.charite.de/genewanderer/

GeneWanderer

Marshfield genetic map, http://www.bli.uzh.ch/BLI/Projects/

genetics/maps/marsh.html

NCBI Map Viewer, http://www.ncbi.nlm.nih.gov/mapview/

The Am

Online Mendelian Inheritance in Man (OMIM), http://www.

omim.org

SMART database, http://smart.embl-heidelberg.de/

UCSC Genome Browser, http://genome.ucsc.edu

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