Human mutations in NDE1 cause extreme microcephaly with lissencephaly [corrected].

Abstract

Genes disrupted in human microcephaly (meaning "small brain") define key regulators of neural progenitor proliferation and cell-fate specification. In comparison, genes mutated in human lissencephaly (lissos means smooth and cephalos means brain) highlight critical regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure, a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the C-terminal NDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulation of cytoskeletal dynamics in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable, cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.

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ARTICLE
Human Mutations in NDE1 Cause Extreme
Microcephaly with Lissencephaly
Fowzan S. Alkuraya,1,8,9,11Xuyu Cai,2,3,11Carina Emery,4Ganeshwaran H. Mochida,2,5,6
Mohammed S. Al-Dosari,1,10Jillian M. Felie,2R. Sean Hill,2Brenda J. Barry,2Jennifer N. Partlow,2
Generoso G. Gascon,7Amal Kentab,9Mohammad Jan,7Ranad Shaheen,1Yuanyi Feng,4,*
and Christopher A. Walsh2,3,5,*
Genes disrupted in human microcephaly (meaning ‘‘small brain’’) define key regulators of neural progenitor proliferation and cell-fate
specification. In comparison, genes mutated in human lissencephaly (lissos means smooth and cephalos means brain) highlight critical
regulators of neuronal migration. Here, we report two families with extreme microcephaly and grossly simplified cortical gyral structure,
a condition referred to as microlissencephaly, and show that they carry homozygous frameshift mutations in NDE1, which encodes
a multidomain protein that localizes to the centrosome and mitotic spindle poles. Both human mutations in NDE1 truncate the
C-terminalNDE1domains, which are essential for interactions with cytoplasmic dynein and thus for regulationof cytoskeletal dynamics
in mitosis and for cell-cycle-dependent phosphorylation of NDE1 by Cdk1. We show that the patient NDE1 proteins are unstable,
cannot bind cytoplasmic dynein, and do not localize properly to the centrosome. Additionally, we show that CDK1 phosphorylation
at T246, which is within the C-terminal region disrupted by the mutations, is required for cell-cycle progression from the G2 to the
M phase. The role of NDE1 in cell-cycle progression probably contributes to the profound neuronal proliferation defects evident in
Nde1-null mice and patients with NDE1 mutations, demonstrating the essential role of NDE1 in human cerebral cortical neurogenesis.
Introduction
The exquisitely organized formation of cerebral cortical
neurons from the cortical neuroepithelium has provided
an important system for studying the control of cell prolif-
eration and cell fate. Cortical progenitors, forming a pseu-
dostratified epithelium with nuclei in the ventricular
germinal zone, have the capacity to undergo symmetrical
cell divisions to form two dividing daughter cells or asym-
metrical cell divisions to generate one dividing daughter
and postmitotic neurons that populate the developing
cortex. After exiting the cell cycle, postmitotic neurons
migrate away from the ventricular zone to the incipient
cerebral cortical layers to establish the highly organized
cortical architecture. Human autosomal-recessive primary
microcephaly (MCPH [MIM 251200]), or microcephaly
vera, manifests as small but architecturally fairly normal
brains. Multiple human genes mutated in MCPH encode
proteins that localize to the centrosome and/or mitotic
spindle poles.1Many of them have also been implicated
inregulatingprogenitorcell-cycleprogressionandindeter-
mining whether progenitors will continue proliferating or
differentiate into postmitotic neurons.1In contrast to
microcephaly, human lissencephaly manifests as a simpli-
fied cortical gyration pattern that reflects abnormal histo-
logical organization of the cortical layers but normal brain
volumeand thusmost often reflectsdisruption ofneuronal
migration.2Identified genetic causes of lissencephaly
include mutations in LIS1 (MIM 601545),3DCX (MIM
300121),4RELN (MIM 600514),5and TUBA1A (MIM
602519).6Recently, mutations in WDR62 [MIM 613583]
have been associated with microcephaly and with a variety
of architectural defects of the cortex,7–9suggesting addi-
tional overlap in the genes that regulate proliferation and
migration. However, there have long been rare cases in
which a reduced brain size typical of microcephaly is
also associated with simplification of cerebral cortical
gyration that falls within the spectrum of lissencephaly.
These two findings together have been referred to as
‘‘microlissencephaly10,11,’’but thegeneticandmechanistic
causes of microlissencephaly are unknown.
Nuclear distribution E (NudE) was originally identified in
Aspergillus nidulans as an essential regulator of a common
nuclear migration pathway, which also involves NudC
(DYNC1H1 [MIM 600112]) and NudF (LIS1).12
mammalian orthologs of NudE include NDE1 (MIM
609449) and NDE1-Like 1 (NDEL1 [MIM 607538]). Nde1
is highly expressed in cortical neural progenitors and
encodes a protein that localizes to the centrosome and
mitotic spindle poles. This protein is also known to
The
1Department of Genetics, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia;2Division of Genetics, Howard Hughes Medical
Institute, and Manton Center for Orphan Disease Research, Children’s Hospital Boston, Boston, MA 02215, USA;3Program in Biomedical and Biological
Sciences, Harvard Medical School, Boston, MA 02215, USA;4Department of Neurology and Center for Genetic Medicine, Northwestern University Feinberg
School of Medicine, Chicago, IL 60611, USA;5Departments of Pediatrics and Neurology, Harvard Medical School, Boston, MA 02215, USA;6Pediatric
Neurology Unit, Department of Neurology, Massachusetts General Hospital, Boston, MA 02114, USA;7Department of Neurology, King Faisal Hospital
and Research Centre, Jeddah 11211, Saudi Arabia;8Department of Anatomy and Cell Biology, College of Medicine, Alfaisal University, Riyadh 11533,
Saudi Arabia;9Department of Pediatrics, King Khalid University Hospital and College of Medicine, King Saud University, Riyadh 11472, Saudi Arabia;
10Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
11These authors contributed equally to this work
*Correspondence: yuanyi-feng@northwestern.edu (Y.F.), christopher.walsh@childrens.harvard.edu (C.A.W.)
DOI 10.1016/j.ajhg.2011.04.003. ?2011 by The American Society of Human Genetics. All rights reserved.
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physically interact with the cytoplasmic dynein complex
and Lis1.13–15The dynein-Lis1-Nde1 complex has an
essential role in the cytoskeleton dynamics of a wide range
of cellular processes, including mitosis, nuclei positioning,
and cell migration.2,16Loss of Nde1 in mouse models
causes profound defects in cerebral corticogenesis but
only modest defects in neuronal migration.14Nde1-null
cortical progenitors showed defects in centrosome duplica-
tion and mitotic-spindle assembly. These mitotic defects
resulted in severe mitotic arrest or delay, spindle misorien-
tation, and mispositioning of the mitotic chromosomes
and were thought to account for the premature depletion
of progenitor cells in Nde1-null brains through impaired
cell-cycle progression and for premature cell-cycle exit by
progenitors to form neurons.14
In this study, we demonstrate that NDE1 mutations
cause a severe microlissencephaly syndrome resembling
that described initially by Norman and Roberts.10,11The
identified frameshift mutations result in truncation of
the C-terminal domains and disruption of several key
functions of NDE1. Additionally, we show that Nde1 is
phosphorylated by Cdk1 and that CDK1 phosphorylation
at T246, within the C-terminal region that is disrupted by
both mutations, is required for cells to progress through
the G2/M phase of mitosis.
Subjects, Material, and Methods
Human Studies
The human studies protocols were reviewed and approved by the
institutional review board of the Children’s Hospital Boston and
the King Faisal Specialist Hospital and Research Centers at Riyadh
and Jeddah and performed in accordance with the ethical stan-
dards. Proper informed consent was obtained. Standard protocols
were used for blood draw and DNA extractions.
Genome-wide Linkage Analysis
We genotypedfamily1by usingtheAffy250K StyI SNPChipas per
the manufacturer’s protocol and family 2 by using the Illumi-
na660W-Quad Chip at the W.M. Keck Foundation Biotechnology
Resource Laboratory at Yale University. We calculated single and
multipoint LOD scores by using Allegro and assuming a recessive
mode of disease inheritance, full penetrance, and a disease-allele
frequency of 0.0001.
Nucleotidenumbersarein
NM_017668.2, where A of the ATG translational start site is
designated as þ1) coordinates, and amino acid numbers are in
reference to protein (RefSeq NP_001137451.1) coordinates, per
HGVS guidelines.
referencetocDNA (RefSeq
Sanger Sequencing
We amplified NDE1 coding exons as well as flanking intronic
sequences by PCR, and then either bidirectionally sequenced
them by using ABI 3730XL DNA Analyzer or submitted them
to Polymorphic DNA Technologies for Sanger capillary electro-
phoresis. See Table S1 for PCR primer sequences and conditions.
Sequencing of more than 200 neurologically normal control
samples and 96 unaffected individuals from Saudi Arabian fami-
lies with unrelated disorders failed to identify either mutant
variant.
Cell Culture, Transfection, Cell Synchronization,
and Flow Cytometry
293T cells were cultured in Dulbecco’s Modified Earle Medium
(DMEM) containing 10% fetal bovine serum (FBS). Mouse embry-
onic fibroblasts (MEFs) were isolated from E13.5 Nde1?/?mice
and their littermates and were cultured in DMEM with 10% FBS
for no more than two passages. We transformed human lympho-
blasts from normal and NDE1?/?individuals and from heterozy-
gous NDE1-/þparents by using Epstein-Barr virus and cultured
them in RPMI medium supplemented with 15% fetal calf serum
(FCS). For FLAG-NDE1 overexpression, we transfected 293T cells
with specified plasmids (empty FLAG-containing vector, FLAG-
NDE1-WT, FLAG-NDE1–p.Pro229TrpfsX85,
p.Leu245ProfsX70) by using Fugene6 (Roche). For Cdk1 and
MAPK knockdown, we cotransfected Silencer Select Pre-Designed
and Validated siRNAs (Applied Biosystems) designed against
Cdk1, MAPK1, and MAPK3 at12 nM with Nde1-WT or Nde1-
T246A plasmids into 293T cells for 48 hr by using lipofectamine
(Invitrogen) as indicated. For cells with enriched G0 and G2/M
populations, 293T cells were starved of serum in DMEM for 48 hr
(G0) and recovered in 10% serum for 3 hr; 0.1 mM nocodazole
was added for 24 hr so that cells would arrest at G2/M. The flow-
cytometry study was performed as previously described;14mitotic
cells were labeled with phospho-histone H3 antibody, and about
60,000 cells were analyzed for each sample.
To measure the mitotic index, we fixed MEFs at passage 1 with
cold methanol, and we identified cells in G2/M by the MPM-2
antibody immunostaining. We counted approximately 1500 cells
from 10 randomly selected fields, and we identified percentages of
MPM-2-positive versus total cells by using Hoechst 33342 stain-
ing. We performed the statistical analysis by using the chi-square
test of homogeneity for two independent samples.
and FLAG-NDE1-
Immunoblotting and Immunoprecipitation
We prepared cell lysates by using either lysis buffer (for immuno-
precipitation) containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl,
0.4% NP-40, 1 mM NaF, 10 mM b-glycero-phosphate, 10 nM
calyculin A, 1 mM Na3VO4, 1 mM PMSF, and a protease inhibitor
cocktail mix (Roche) or (for direct immunoblotting) RIPA buffer
(50 mM Tris-HCl 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, and 0.1% SDS) containing a protease inhibitor
cocktail mix (Roche). We normalized protein concentrations by
using the bicinchoninic acid assay (Thermo Scientific). For immu-
noprecipitation, cell lysates were incubated with anti-FLAG M2
affinity gel (Sigma) for 2 hr at 4?C and then washed three times
with the same lysis buffer. Protein samples were then eluted
with 3XFLAG peptides (150 ng/ml) for 1 hr at 4?C. For immuno-
blotting, cell lysates or immunoprecipitation elution were
boiledin LDS sample buffer (Invitrogen) and then electrophoresed
on 4%–12% Bis-Tris or 7% Tris-Acetate SDS-PAGE (Invitrogen)
and transfered onto a PVDF membrane (Millipore). We blocked
membranes for 30 min in TBST that contained 5% nonfat
milk or Odyssey Blocking Buffer (LICOR Biosciences) at room
temperature, incubated them with primary antibodies according
to the antibody manufacturer’s instructions, and then incubated
them with HRP-conjugated secondary antibodies (Cell Signaling
Technology) or fluorescent-dye-conjugated secondary antibodies
(LICOR Biosciences).Immunosignals weredetectedby
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537

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SuperSignal West Pico Chemiluminescent (Pierce) or the Odyssey
Infrared Imaging System (LICOR Biosciences).
To create a phospho-specific antiserum against T246 of NDE1,
we coupled a synthetic peptide corresponding to amino acids
R234–T246 of Nde1 that includes a phosphorylated T246 residue
(p-Nde1T246) to KLH and used it to immunize rabbits via standard
procedures provided by Covance. The resulting antisera were
purified against Affi-Gel 10 and 15 columns (Biorad) to which
the p-Nde1T246 peptide was coupled. The affinity-purified
p-Nde1T246peptideantiserawerefurtherpurifiedthroughacolumn
coupled with unphosphorylated Nde1T246 peptide to deplete
immunoreactivities against unphosphorylated proteins. The rabbit
antibody against total Nde1 was used as previously described.14
In addition, the following antibodies were purchased and used
accordingtothemanufacturer’sinstructions:mouseFLAG(M2)anti-
body and anti-FLAG beads (Sigma), rabbit anti-DYKDDDDK (FLAG)
(Cell Signaling Technology), rabbit anti-LIS1 (Bethyl Laboratory),
mouse monoclonal anti-dynein intermediate chain (clone 74.1)
(Millipore), rabbit anti-dynein heavy chain (Santa Cruz Biotech-
nology), mouse monoclonal anti-Cdk1 (Millipore), rabbit anti-p42/
44 MAP kinase (Cell Signaling Technology), and mouse anti-
MPM-2 (Upstate). Immunohistochemistry and immunocytochem-
istry protocols are described in detail elsewhere.14
For the FLAG-NDE1 construct,
NM_017668.2, starting from ATG) was amplified by PCR and
subcloned into the p3XFLAG-CMV-10 vector (Sigma); the two
mutant constructs were generated by site-directed mutagenesis
so that 684_685AC (c.684_685del; p.Pro229TrpfsX85) was deleted
or 733C (c.733dup; p.Leu245ProfsX70) was duplicated, respec-
tively. Nde1-T246A construct was generated by site-directed muta-
genesis on Nde1-WT cDNA construct as described previously.14
NDE1
cDNA(RefSeq
In Vitro Cdk1/Cdc2 Kinase Assay
Myc-tagged Nde1 and Nde1 point mutants were expressed in 293T
cells and immunoprecipitated with the 9E10 Myc monoclonal
antibody. Recombinant cyclin B1 and Cdk1/Cdc2 were purchased
from New England Biolabs, and the kinase assay was performed
with the reaction buffer provided by the manufacturer. After the
immunocomplexes were washed and equilibrated to the kinase
reaction buffer, they were incubated with 0.1 unit of Cdk1/Cdc2
kinase and 50 mM [g?32P] ATP (specific activity of 100 mCi/mmol)
at room temperature for 10 min. The reaction products were
analyzed by 10% SDS PAGE followed by autoradiography.
Results
Clinical Characterization of Microlissencephaly
Ongoing efforts to characterize the genetic bases of
microcephaly by a large collaborative effort known as the
Microcephaly Collaborative (Table S2, available online)
have identified two families whose children share remark-
ably severe microcephaly (head circumferences more than
11 and 13 standard deviations [SD] below the mean for
age), abnormal cortical gyration, short length (more than
2 and 4 SD below the mean for age), microsomia (weight
more than 2 and 5 SD below the mean for age) and a prom-
inent broad nasal bridge (Figure 1); both families have
been approved by the institutional review board.
Family 1 originates from eastern Saudi Arabia. The
parents are healthy and are reported to be first cousins.
They have two daughters affected by extreme micro-
cephaly(Figure 1A).The
(08DG00535; family1, IV-1 in Figure 1A) was first evalu-
ated at the age of 33 months, at which time her head
circumference was 34.4 cm, length was 81.4 cm, and
weight was 8.6 kg (8.9, 3, and 3.8 SD below the mean,
respectively). Physical examination at that time was
notable for marked hypertonia and global developmental
delay. Magnetic resonance imaging (MRI) scans revealed
severe microcephaly with a proportionate reduction in
the size of most other brain structures, including the cere-
bellum and brain stem, associated with agenesis of the
corpus callosum. The gyral folding of the cerebral cortex
was extremely simplified; there were almost no detectable
sulci other than the Sylvian fissure. At the age of 7 years,
she displayed extreme microcephaly (the head circumfer-
ence of 35 cm was 13.4 SD below the mean) and evidence
of retarded growth; she had a length of 105 cm and weight
of 10 kg (3.1 and 4.7 SD below the mean, respectively). She
remained seizure-free but was only able to roll over, and
her social and cognitive development was limited to spon-
taneoussmiling. The second
(08DG00536; family 1, IV-2 in Figure 1A) was first evalu-
ated at birth, at which time her length and weight were
normal, head circumference was 26.5 cm (5.6 SD below
the mean), and anterior fontanelle was almost closed.
Her neurological course was static and characterized by
marked hypertonia; she gained virtually no milestones
beyond spontaneous smiling and rolling over by the age
of 5.5 years but developed no seizures. At that time her
head circumference was 34 cm, length was 85 cm, and
weight was 8.9 kg (12.7, 5.5, and 4.8 SD below the mean,
respectively). Laboratory investigations, including plasma
acylcarnitines, carnitine, amino acids, urine organic acids,
ammonia, lactic acid, and high-resolution karyotype, were
all normal. MRI scans showed microcephaly, severe simpli-
fication of the gyral pattern (lissencephaly), agenesis of the
corpus callosum, and colpocephaly (enlargement of the
posterior lateral ventricles, often associated with corpus
callosum defects), features that have been described in
microlissencephaly (Figures 1B–1G; see Movies S1–S6).
Family 2 originates from western Saudi Arabia. The
parents are healthy and are reported to be first cousins.
They have a daughter and son with extreme microcephaly
and two healthy sons and had three additional pregnan-
cies that resulted in spontaneous abortions (Figure 1A).
The affected daughter was reported to have microcephaly
without seizures, but no additional information was
available (family 2, II-4 in Figure 1A). The affected son
(MC-14901; family 2, II-7 in Figure 1A) displayed micro-
cephaly and dysmorphic features at birth, but growth
parameters were not available. A head CT at that time
revealed small brain size. Seizures began at two months
of age and were described as starting on the left side and
progressing to full-body convulsions with up-rolling of
the eyes. He was first evaluated at 9 months of age, at
which time he could not roll or control his head, and
first affected daughter
affecteddaughter
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a neurologic exam revealed increased reflexes, decreased
tone, normal power, and positive Babinski signs. MRI scans
at 11 months of age showed a marked decrease in the size
of both cerebral hemispheres, a large midline fluid-filled
structure, dilatation of the right lateral ventricle, a small
cerebellum, and agenesis of the corpus callosum. The
thalami were not fused, and a midline falx was noted.
Laboratory investigations revealed a normal karyotype
(46, XY), an unremarkable acylcarnitine profile, and
normal amino acid analysis by tandem mass spectrometry.
An evaluation at 3.5 years of age recorded his head circum-
ference as 32 cm (11.1 SD below the mean), length as
88 cm (2.8 SD below the mean), and weight as 13 kg
(6th percentile).
Identification of Homozygous NDE1 Mutations
All patients were born to consanguineous marriages, sug-
gesting autosomal inheritance and allowing homozygosity
mapping,whichidentifiedonlyonecommonhomozygous
region larger than 1Mb—at chromosome 16p13.11—
shared by all three affected children (DNA from the second
affected child in family 2 was unavailable). The maximal
LOD scores of the two pedigrees at the homozygous region
are 1.8 and 1.2, respectively. The homozygous locus is 4.6
Mbinsizeandcontainsapproximately35annotatedgenes,
including NDE1 (Figure 2A). NDE1 was selected as the top
candidate given that the Nde1 mutations strongly affected
neural progenitor proliferation and that defects of both
proliferation and neuronal migration were observed in
mice deficient for both Nde1 and Lis1.14,17,18Direct
sequencing of NDE1 revealed frameshift mutations in
both families (Figures 2B and 2C). Family 1 showed
a c.684_685del mutation in exon 6 that creates a transla-
tional frameshift at codon 229 of the normal 335 amino
acid protein sequence (p.Pro229TrpfsX85) and predicts
aproteinthatconsistsof312aminoacids,terminatingafter
Family 2
08DG0053508DG00536
A
B
Normal
(2 years)
08DG00536
(4.5 years)
CD
E
F
G
Family 1
I
II
III
IV
First
cousins
MC-14901
I
II
1*
1*
1
1*
2*
2*
2* 2*
23456
7*
7*
Figure 1.
(A) Both families are from Saudi Arabia. Parents of family 1 are first cousins and have two affected female children (08DG00535, IV-1 and
08DG00536, IV-2). Whole-blood DNA from both parents and both affected children was obtained and analyzed (indicated by an
asterisk). Parents of family 2 are first cousins who had seven reported pregnancies, producing one affected male (MC-14901, II-7),
one affected female (II-4) (not available for analysis), two unaffected males, and three pregnancies that resulted in fetal demise.
Whole-blood DNA from both parents and the affected male (MC-14901, II-7) was obtained and analyzed (indicated by an asterisk).
(B–G)Representative MRIimagesof 08DG00536 (IV-2) from family 1 at 4.5years ofage, demonstrating thedrastic reduction in brainsize
(E–G), agenesis of the corpus callosum, and abnormal gyral pattern compared to that seen in a normal 2-year-old child (B-D). Sagittal T1
(B, C, E, and F) and axial T2 (D and G) sections are shown. The scale bar represents 5 cm. Additional imagesare available as Movies S1–S6.
Pedigrees and Radiographic Features of the Two Consanguineous Families with Microlissencephaly
The American Journal of Human Genetics 88, 536–547, May 13, 2011
539

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c.684_685del
c.733dup
LIS1
CENP-F
Dynein
Centrosomal localization
p.Leu245ProfsX70
p.Pro229TrpfsX85
B
A
C
08DG00536
PtdelAC
Control
Control
Scale
chr16:
1 Mb
13500000 14000000
ERCC4
BC039386
14500000
PARN
15000000
NPIP
NTAN1
RRN3
15500000
MPV17L
KIAA0430
16000000
C16orf63
16500000
AK310228
17000000
FLJ11151
CR749545
AK000877 MKL2
CR593775
BFAR
PLA2G10
NPIP
NPIP
URG7
NOMO1
FLJ00322
PDXDC1
LOC728138
AK125313
DQ596229
NPIP
FLJ00285
Nbla00537
C16orf45
NDE1
MYH11
ABCC1
ABCC6
NOMO3
FLJ00322
LOC339047
LOC339047
XYLT1
AX747757
LOD score
Scale
chr16:
08DG00535_Hz16
08DG00536_Hz16
MC-14901_Hz16
10 Mb
500000010000000150000002000000025000000 30000000
chr16 16p13.3p13.2p12.3 16p12.116p11.2p11.1 q11.216q12.112.21316q21q22.1 22.3 q23.124.1
0 50001000015000 2000025000 30000
335
313
312
LIS1
frameshift
LIS1
frameshift
Pt
MC-14901
dupC
1234567 8 9
p.Pro229TrpfsX85
p.Leu245ProfsX70
Figure 2.
(A) Homozygosity analysis of both pedigrees identified a 4.6 Mb region on chromosome 16 that is homozygous in all three affected
children. All homozygous SNPs are represented as blue, and heterozygous SNPs are represented as yellow. The maximal LOD scores
of the two pedigrees at the homozygous region are 1.8 and 1.2, respectively. The shared region of homozygosity contains approximately
35 annotated genes, including NDE1.
(B) Human NDE1 consists of nine exons (eight coding exons), which encode a protein with 335 amino acids harboring multiple protein-
interaction domains. Two frameshift mutationswere indentified in exons 6 and 7, respectively; both are predicted to disrupt the CENP-F,
dynein interaction domain, centrosomal localization domain, and at least two conserved phosphorylation residues implicated in
Two NDE1 Mutations Identified within an Overlapping Region of Homozygosity in Both Pedigrees on Chromosome 16
540
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the addition of 84 abnormal amino acids (Figures 2B and
2C). Exon 7 of family 2 showed a c.733dup mutation that
creates a frameshift mutation at codon 245 (into the same
abnormal reading frame as the mutation in family 1).
This frameshift is predicted to result in a protein that is
truncated after the addition of 69 abnormal amino acids
(p.Leu245ProfsX70). Both mutations were homozygous
in affected individuals; segregated perfectly with disease
in each family; were absent from more than 200 normal
individuals, including 96 Saudi Arabian controls; and
were not seen in the 1000 Genomes Project, all of which
strongly suggests that they are bona fide mutations. This
conclusion was further supported by identification of addi-
tional NDE1 alleles in patients sharing similar clinical
features by Bakircioglu et al.19in this issue.
Characterization of the Mutant NDE1 Proteins
On the basis of data from NDE1 and its paralog, NDEL1, it
appears that the truncated NDE1 proteins, if stable, would
lack critical protein domains (Figure 2B). The C terminus of
NDE1 includes a domain required for interaction with
CENP-F, which directs NDE1 to kinetochores,20and
another domain that regulates binding to the cytoplasmic
dynein complex21,22and to Su48,23which was recently
found to regulate centrosomal localization of NDE1.
Although RT-PCR with patient lymphoblasts from family
1 suggested an equal abundance of NDE1 transcripts
between patients and controls (data not shown), immuno-
blot analysis of patient lymphoblasts showed no detect-
able NDE1 protein in the two affected patients (family 1,
IV-I and IV-II) carrying the p.Pro229TrpfsX85 mutation
(Figure 3A), whereas the heterozygous parents showed an
~50% reduction in protein levels. These data suggest that
the frameshift mutation caused instability of the protein
and subsequent degradation. Thus, the severe reduction
in brain volume in these patients is far more severe than
the dramatic ASPM-associated microcephaly24–26
appears to be associated with loss of the NDE1 protein,
although some mutant protein in some tissues cannot be
ruled out.
In order to further study the functional role of the
C terminus of NDE1, we engineered cDNAs corresponding
to the p.Pro229TrpfsX85 and p.Leu245ProfsX70 alleles
and fused them with FLAG tag (Figure 2B). Despite the
possibleproteininstability
proteins, overexpression allowed recovery of enough
protein so that the effects of the two mutations on NDE1
binding to LIS1 and to dynein could be studied. Wild-
type FLAG-NDE1 can be easily coimmunoprecipitated
with the dynein complex (Figure 3B), and both frameshift
mutants completely abolished dynein binding (Figure 3C).
NDE1 also binds LIS1 but through the coiled-coil domain
and
ofendogenousmutant
at the amino terminus of NDE1,13,27and LIS1 binding
was normal or even enhanced (Figure 3C) in the FLAG-
tagged mutant proteins. Because during neurogenesis
the cytoplasmic dynein complex has profound roles,
including mitoticspindle
nuclear migration, and neuronal migration, the loss of
an important dynein regulator could impact multiple
aspects of neurogenesis.
Both mutations are also predicted to abolish the centro-
somal localization domain located at the C terminus of
NDE1 (Figure 2B). To examine the effects of mutations
on NDE1 subcellular localization, we transfected GFP-
tagged wild-type or p.Leu245ProfsX70 mutant NDE1 in
293T cells. Wild-type GFP-NDE1 localized to the centro-
some, consistent with previous findings (Figure 3D),
whereas mutant GFP-NDE1-p.Leu245ProfsX70 failed to
target thecentrosome but either presented as noncentroso-
mal aggregates or presented diffusely in the cytoplasm.
(Figures 3E and 3F). Similar results were reported by Bakir-
cioglu et al.19after analyzing the p.Pro229TrpfsX85 allele.
Therefore, both NDE1 mutations disrupt at least two key
functions of NDE1, suggesting that the developmental
defects seen in the patients are likely caused by the loss
of NDE1 function.
organization,interkinetic
Phosphorylation by Cdk1 at Nde1 T246 Regulates
G2/M Transition
The p.Pro229TrpfsX85and
also eliminate key potential phosphorylation sites in the
C terminus of NDE1. We previously reported that NDE1
redistributes when cells enter mitosis.14In addition,
Nde1 and its paralog, Ndel1, have been reported as mitotic
phospho-proteins.28Phosphorylation of NDEL1 by the
mitotic kinases Cdk1/Cdc2 and Aurora-A are involved in
cell-cycle-dependent events such as centrosome matura-
tion, G2/M transition, and microtubule remodeling during
mitosis.2,29,30We confirmed that NDE1 and Nde1 are also
regulated by mitotic phosphorylation; NDE1 extracted
from mitotically arrested cells (G2/M) often appeared in
slowly migrating forms on immunoblots compared to
the NDE1 from cells arrested at G0 in serum-free media;
this suggests increased phosphorylation (Figure 4A). Scan-
site analysis31identified putative Cdk1/Cdc2 phosphoryla-
tion sites on NDE1 that are conserved in NDEL130,32,33
(Figure 4B), whereas threonine 246 of NDE1 and Nde1
is predicted as the most favorable Cdk1 target at high
stringency by Scansite, is the most conserved site across
species (Figure 4B), and is the only site removed by both
mutations. To investigate the potential regulation of
Nde1 by mitotic phosphorylation, we generated an
antiserum that specifically recognizes phospho-T246 of
Nde1 (p-Nde1T246). The slowly migrating Nde1 band
p.Leu245ProfsX70 alleles
mitotic progression (T246 and S250) at the C terminus. The black bars show noncoding exons; open bars show coding exons. The red
dots indicate potential phosphorylation sites.
(C) Representative Sanger sequencing traces indicating the two base-pair deletions identified in patient 08DG00536 (family 1, IV-2 in
Figure 1A) and the one base-pair duplicated identified in patient MC-14901 (family 2, II-7 in Figure 1A).
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seen in G2/M-arrested cells is specifically recognized by
this phospho-T246 antiserum, confirming that NDE1 is
phosphorylated at T246 at G2/M. Immunofluorescence
studies with the same antiserum showed that it recognizes
cells that appear to be at G2/M (Figure 4C) and highlighted
metaphase neocortical apical progenitors along the sur-
face of lateral ventricles in mouse developing brains
(Figure 4D).
To test whether T246 of Nde1 is a Cdk1/Cdc2 target, we
showed that knockdown of Cdk1 specifically abolished the
p-Nde1T246 immunosignal and left the total Nde1 protein
level unchanged (Figure 4E). Although the flanking
sequence of Nde1 T246 also matches a preferred Erk1/2
phosphorylation site, PXTP, knockdown of Erk1 (MAPK3)
or Erk2 (MAPK1) showed little effect on p-NdelT246 levels
(Figure 4E), suggesting that NDE1 T246 is not a preferred
target of MAPK1 or MAPK3 in vivo. Furthermore, we
showed that recombinant Cdk1 was able to phosphorylate
Myc-Nde1 that had been immunoprecipitated in vitro
(Figure S1). Interestingly, mutating T215, T246, or
both into phosphorylation-deficient residues (p.T215A,
p.T246A, and p.T215/T246A, respectively) reduced but
did not abolish the32P signal; this suggests that additional
sites on Nde1 are also phosphorylated by Cdk1 in vitro.
Finally, to test whether the phosphorylation of T246
is functionally important, we overexpressed the Nde1-
T246A mutant and found that cells expressing this T246
phosphorylation-deficient mutant arrest at the G2 phase
with increased 4N DNA content but with reduced pH3þ
staining, a marker that identifies the mitotic population
(Figure 4F). These data suggest that phosphorylation of
T246 is essential for the G2/M transition and is consistent
IB: FLAG-
NDE1
IB: LIS1
IP: FLAG
Lysates
IB: DIC
WT
Ctrl
A
Actin
IB:
NDE1
IB:
Total Lysate
- + - +
IP: FLAG
IB: DIC
IB: DHC
FLAG-
NDE1
C
B
D
IB: FLAG-
NDE1
IB: LIS1
IB: DIC
08DG00535
(IV-1)
08DG00536
(IV-2)
Father
(III-1)
Mother
(III-2)
Wildtype ctrl
Family 1
EGFP-NDE1-WT
EGFP-NDE1-p.Leu245ProfsX70
Merge EGFP-NDE1Pericentrin
E
F
p.Leu245
ProfsX70
p.Pro229
TrpfsX85
Figure 3.
Are Unstable and Lack Critical Functions
(A) NDE1 protein was undetectable in the
whole-cell lysates collected from lympho-
blasts of both patients (IV-1 and IV-2 in
Figure 1A) in family 1 harboring the
c.684_685del mutation. The protein levels
of NDE1 from the parents (III-1 and III-2 in
Figure 1A) were reduced by roughly 50% in
comparison to the wild-type control; this
is consistent with their known heterozy-
gous carrier status. Immunoblotting (IB)
was performed with an antibody against
NDE1 and actin as a loading control.
(B) Wild-type FLAG-NDE1 interacts with
the dynein complex. Immunoprecipita-
tion was performed with anti-FLAG M2
beads.Cells transfected
3XFLAG-CMV vector were labeled as ‘‘?’’
and used as the negative control; cells
transfected with wild-type FLAG-NDE1
vector were labeled as ‘‘þ.’’ The following
abbreviations are used: DIC, dynein inter-
mediate chain 74.1; DHC, dynein heavy
chain.
(C) The interaction with the dynein
complex was abolished in both mutant
NDE1 proteins,which confirmsthedisrup-
tion of the dynein-binding domain by
both mutant alleles. However, the interac-
tion with LIS1 was preserved or even
slightlyenhanced
proteins.
(D–F) The centrosomal localization of
NDE1 was abolished in the mutants.
(D) Wild-type EGFP-NDE1-WT was trans-
fected into 293T cells and localized to
the centrosome. (E and F) Mutant EGFP-
NDE1-p.Leu245ProfsX70 was transfected
into 293T cells and did not localize to
the centrosome. It either formed noncen-
trosomalaggregates
diffusely in the cytoplasm. (F) Centro-
somes labeled by pericentrin are indicated
by the arrowheads and magnified in the
upper left insets. The scale bar indicates
10 mm.
The Mutant NDE1 Proteins
withempty
inbothmutant
(E)orlocalized
542
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Page 8

with its role as a target of Cdk1. These data indicate that
Nde1 T246 is an essential substrate for Cdk1 and provide
a mechanistic explanation for how the removal of the
C terminus of Nde1, or loss of the entire Nde1 protein,
blocks cell-cycle progression, which would account for
the extreme microcephaly caused by NDE1 mutations.
Loss of NDE1 or Nde1 Disrupts Mitotic Progression
in Both Human and Mice
Mouse embryonic fibroblasts with Nde1 mutations (Figures
5A–5D)showeddefectsinmitoticprogression,asevidenced
by an increased mitotic index; abnormal spindle structures
such as multipolar spindles (Figure 5B); and chromosome
Figure 4.
(A) Nde1 is phosphorylated during mitosis. Immunoblotting of Nde1 with total and phospho-specific Nde1 antibody from serum-
starved G0 and mitoticallyarrested (G2/M) 293Tcells and Nde1?/?cells (lanes 1, 2, and 3, respectively). A band of slowly migratingphos-
phorylated Nde1 (p-Nde1) is significantly elevated in the G2/M cell population (lane 2) and absent in the Nde1?/?-negative control (lane
3), indicating the phospho-Nde1 antibody specificity.
(B) Four out of five putative phosphorylation sites previously identified in Ndel1 are conserved in Nde1(T215, T228, T242, and T246).
Among them, T246 and its flanking sequence are the most conserved and represent the only site disrupted in both mutant alleles. Both
mutations also disrupt a potential AurA phosphorylation site (S250), on the basis of its sequence homology to Ndel1.
(C) Phospho-specific antibody against Nde1 T246 exclusively recognizes G2- and M-phase MEFs (upper panel). (Lower panels) Represen-
tative images of a mitotic cell (marked in square) and an interphase cell (marked by arrowhead). The scale bar indicates 10 mm.
(D) Immunohistological staining of the cerebral cortical ventricular zone from an E14.5 mouse brain with p-Nde1T246 antibody (red)
and b-catenin (green) shows preferential staining of M-phase cell along the ventricular surface. The scale bar indicates 50 mm. The
ventricular zone (VZ) and subventricular zone (SVZ) are indicated at their corresponding position in the image.
(E) Nde1 was phosphorylated at T246 by Cdk1 but not by MAPK1 or MAPK3. Two nanomoles of Silencer Select Pre-Designed and
Validated siRNA to Cdk1, MAPK1, and MAPK3 siRNA (Ambion/Applied Biosystems) was cotransfected with 1 mg Nde1-WT/T246A
plasmid into 293T cells. Immunosignals were abolished specifically by Cdk1 siRNA or by the Nde1-T246A point mutation but not by
siRNA against MAPK1 or MAPK3.
(F) Cell-cycle profiles of 293T cells transfected with GFP, GFP-Nde1, and GFP-Nde1-T246A. An increased 4N (G2/M) population was
observed by GFP-Nde1-T246A overexpression, suggesting the important role of p-Nde1T246 in cell-cycle progression. In addition,
flow cytometry on the 4N population by phospho-histone H3 (pH3, mitotic marker) reveals that there are a decreased number of mitotic
cells in the GFP-Nde1-T246A transfected cells despite the more abundant 4N population, suggesting that a large number of these cells
arrested in the G2 phase. The y axis represents the ratio of pH3(þ) to pH3(?) cells.
T246 of Nde1 is Phosphorylated by Cdk1 and Is Involved in the G2/M Transition
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543

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Figure 5.
Mitotic Index and Abnormal Mitotic Spindles
(A–D) Early-passage (P0-3) primary MEFs derived
from wild-type (Nde1þ/þ) and mutant (Nde1?/?)
embryoswereanalyzed.
increased by approximately 50% in Nde1?/?
MEFs compared to that in Nde1þ/þMEFs at
passage 1 (p value < 0.01, chi-square test of
homogeneity for two independent samples; error
bar represents standard error of the mean). (B–D)
Primary MEFs derived from wild-type (Nde1þ/þ)
and mutant (Nde1?/?) embryos were analyzed
directly for the structure of mitotic spindles by
staining with monoclonal antibody for tubulin
(in green) and Hoechst for chromosomal DNA
(in blue). (B) A normal Nde1þ/þM-phase cell.
(C) An abnormal Nde1?/?M phase cell with tripo-
lar mitotic spindle and misaligned mitotic chro-
mosomes and (D) an abnormal Nde1?/?mitotic
cell with discordant mitotic spindle and chromo-
some alignments. The scale bar indicates 10 mm.
(E–H) NDE1 patient lymphoblasts exhibit similar
defects in mitotic spindle organization. (E and F)
Control lymphoblasts showed normal looking
nuclei and normal a-tubulin staining. (E) An
example of a normal NDE1þ/þinterphase cell.
(F) An example of a normal NDE1þ/þM phase
cell. (G and H) Patient lymphoblasts showed
condensed/fragmentized nuclei and disorganized
a-tubulin. (G) An example of an abnormal
NDE1?/?interphase cell with nuclear fragmenta-
tion and (H) an exampleofan abnormalNDE1?/?
M phase cell with multipolar and disorganized
spindles. The scale bar indicates 10 mm. The
percentage of mitotic cells found with abnormal
spindles was 7%in
and compared to 0% in control lymphoblasts
(p < 0.02, two-tailed chi-square test).
NDE1 Deficiency Leads to Increased
(A)Mitoticindex
patient lymphoblasts
544
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misalignment
lymphoblast cells (Figures 5E–5H) also show spindle-struc-
turedefects,includingtripolarspindles,misalignedmitotic
chromosomes, nuclear fragmentation, and abnormal
microtubule organizations, further supporting the idea
that NDE1 is essential for normal mitotic spindle function.
Patientlymphoblastsalsoshowedexcessivecelldeath(data
not shown), which could also contribute to the develop-
mental defects. We previously reported that the N-terminal
self-association domain of Nde1 is required for proper
mitotic spindle assembly and mitotic progression, which
presumably accounts for the mitotic defects in NDE1- and
Nde1-null cells.14The interesting contrast between the G2
arrest induced by Nde1-T246A overexpression and the M
phase arrest resulting from Nde1 depletion presumably
reflectsthepleiotropicrolesofNde1incell-cycleregulation
and is discussed in greater detail below.
(Figure5C). Similarly,patient-derived
Discussion
Our data indicate that loss of NDE1 produces profound
defects in cerebral cortical size and organization as well
as less profound defects in somatic size. Both of the identi-
fied NDE1 mutations truncate the C terminus of the NDE1
protein, preventing normal dynein binding. Whereas the
overexpressed proteins are somewhat unstable, they retain
LIS1-binding activity and so in principle could act as domi-
nant-negative proteins. However, the absence of a geneti-
cally dominant phenotype, the absence of detectable
mutant proteins in patient cells, and the similarity of the
phenotype in the three patients described here (as well as
additional patients and an additional mutant allele
described by Bakircioglu et al.19), all argue strongly that
the mutations act as simple null alleles.
The C terminus of NDE1 is also mutated by the recurrent
16p inversions that are associated with acute myelogenous
leukemia (AML), which disrupts exon 8 of NDE1, encoding
the extreme C terminus of the protein.34Existing pedigree
analysis and medical follow-up of families 1 and 2,
although somewhat limited, has so far not suggested the
presence of AML or other leukemia in homozygous or
heterozygous carriers of NDE1 mutations, though more
extensive analysis of additional families is needed. On
the other hand, our biochemical analysis suggests that
the AML disruptions, if they create a stable truncated
protein, could potentially create abnormal proteins with
preserved LIS1 binding but abnormal dynein binding,
which might contribute to leukemogenesis.
Whereas mice lacking Nde1 show about a one-third
reduction in brain mass,14neuronal migration is only
moderately deficient, but human NDE1 mutations cause
adecreaseofmorethan50%incorticalvolumeandstriking
architectural disturbances that suggest severely abnormal
neuronal migration (see also Bakircioglu et al.19). The
more striking brain defects seen in humans harboring
NDE1 mutations, especially the marked architectural
defects, could reflect morphological or quantitative defects
in the radial glial cells that normally act as guides for
migrating neurons, or they could reflect the larger human
brain and its increased opportunities for defects in neuro-
genesis. Alternatively, the differences could reflect a greater
role for NDE1 in migrating neurons in humans than in
mice, in parallel to the much more profound defects in
neuronal migration in humans than in mice after removal
of one allele of LIS1. The Nde1 paralog, Ndel1, is highly
similarstructurallyto Nde1, morehighly expressedinpost-
mitotic neurons, and appears to have larger roles in cell
proliferationoutside the brain,mitoticspindle orientation,
and neuronal migration.16,27,29,33,35–38
potential genetic redundancy between NDE1 and NDEL1
could also contribute to the differences between mice and
humans. The overlapping functions of NDE1 and NDEL1
could also relate to distinct cellular phenotypes, namely
interferencewithnormalphosphorylationofNDE1,which
arrests cells in G2 phase, versus simple loss of NDE1, which
arrests cells in M phase. Perhaps redundant functions of
NDEL1 allow NDE1-deficient cells to traverse G2, whereas
lack of normal NDE1 phosphorylation might induce
abnormal protein complexes that interfere with both
NDE1 and NDEL1 function, preventing transit through
mitosis.
Humans with NDE1 mutations show modestly reduced
height and weight as well as cerebral cortical size, though
the defect in head circumference (typically 10 to 14 SD
belowthemean)wasmoremarkedstatisticallythandefects
in height and weight (typically 2 to 5 SD below mean). The
decreased body size could reflect roles of NDE1 in other
tissues, although more specific hypothalamic defects or
nutritional explanations related to the patients’ poor
neurological function cannot be ruled out. Mice with
Nde1 mutations show at most a slight, statistically insignif-
icant reduction in body mass.14Because NDE1, like many
othermicrocephalygenes,isexpressedinmanydeveloping
tissues,13themoresevereinvolvementofthebrainisgener-
allyregardedasreflectingitsmorelimitedabilitytoregulate
itssize,giventhatmostbraincellsarepostmitotic,butother
mechanisms might also contribute to this tissue specificity.
Finally, although analysis of archived DNA samples from
the original microlissencephaly family studied by Norman
and Roberts11did not reveal a detectable mutation in
NDE1 (data not shown), the similarity of the NDE1 pheno-
type to the Norman Roberts syndrome is striking, and
some other cases of microlissencephaly also do not show
NDE1 mutations. Hence, this classical form of microlissen-
cephaly might ultimately reflect defects in proteins that
function in close concert with NDE1 in this highly
conserved centrosomal pathway.
The details of
Supplemental Data
Supplemental Data include one figure, two tables, and six movies
and can be found with this article online at http://www.cell.com/
AJHG/.
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545

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Acknowledgments
We are grateful to the families, clinicians, and researchers who
contributed to this study, including reviews of the MRI findings
by Bernard Chang and Annapurna Poduri. We thank members of
the Microcephaly Collaborative (Table S2) for contributing patient
samples not directly used in this study. F.S.A., G.H.M, and C.A.W.
are supported by a Collaborative Research Grant from the Dubai-
Harvard Foundation for Medical Research. F.S.A. is also supported
by the King Abdulaziz City for Science and Technology (Grant
10-MED941-20/Saudi Arabia). X.C. is supported by the National
Institute of General Medical Sciences (T32 GM007726-35).
G.H.M.wassupportedbyNationalAllianceforResearchonSchizo-
phrenia and Depression as a Lieber Young Investigator. Y.F. is sup-
ported by the NICHD (R01HD56380), and the Schweppe Founda-
tion. C.A.W is supported by the NINDS (RO1 NS 32457) the
Fogarty International Center (R21 NS061772), the Manton Center
for Orphan Disease Research, the National Library of Medicine
Family Foundation, and the Simons Foundation. Genotyping at
the Center for Inherited Disease Research is funded through
a federal contract from the National Institutes of Health (NIH) to
The Johns Hopkins University (HHSN268200782096C and NIH
N01-HG-65403). Genotyping at Children’s Hospital Boston is sup-
portedbytheIntellectualandDevelopmentalDisabilitiesResearch
Centers(P30HD18655).SNPgenotypingoffamily2wasperformed
through the NIH Neuroscience Microarray Consortium. Genotyp-
ing at the Broad Institute is supported by the National Human
Genome Research Institute. C.A.W. is an Investigator of the Ho-
ward Hughes Medical Institute.
Received: January 22, 2011
Revised: March 25, 2011
Accepted: April 7, 2011
Published online: April 28, 2011
Web Resources
The URLs for data presented herein are as follows:
1000 Genomes Project, http://www.1000genomes.org/
GenBank, http://www.ncbi.nlm.nih.gov/Genbank/
HGVS Guidelines, http://www.hgvs.org/rec.html
Online Mendelian Inheritance in Man (OMIM), http://www.
omim.org
Scansite, http://scansite.mit.edu
UCSCGenomeBrowser,http://genome.ucsc.edu/cgi-bin/hgGateway
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