Please cite this article in press as: Li et al.

, Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

REPORT
Biallelic Mutations in Citron Kinase
Link Mitotic Cytokinesis to Human
Primary Microcephaly
Hongda Li,1,2 Stephanie L. Bielas,1,2,3 Maha S. Zaki,4 Samira Ismail,4 Dorit Farfara,1,2 Kyongmi Um,1,2
Rasim O. Rosti,1,2 Eric C. Scott,1,2 Shu Tu,5 Neil C. Chi,5 Stacey Gabriel,6 Emine Z. Erson-Omay,7
A. Gulhan Ercan-Sencicek,7 Katsuhito Yasuno,7 Ahmet Okay Cag layan,8 Hande Kaymakcalan,9
Barsx Ekici, Kaya Bilguvar, Murat Gunel, * and Joseph G. Gleeson1,2,*
9 7 7,

Cell division terminates with cytokinesis and cellular separation. Autosomal-recessive primary microcephaly (MCPH) is a neurodevelop-
mental disorder characterized by a reduction in brain and head size at birth in addition to non-progressive intellectual disability. MCPH
is genetically heterogeneous, and 16 loci are known to be associated with loss-of-function mutations predominantly affecting centro-
somal-associated proteins, but the multiple roles of centrosomes in cellular function has left questions about etiology. Here, we identified
three families affected by homozygous missense mutations in CIT, encoding citron rho-interacting kinase (CIT), which has established
roles in cytokinesis. All mutations caused substitution of conserved amino acid residues in the kinase domain and impaired kinase ac-
tivity. Neural progenitors that were differentiated from induced pluripotent stem cells (iPSCs) derived from individuals with these mu-
tations exhibited abnormal cytokinesis with delayed mitosis, multipolar spindles, and increased apoptosis, rescued by CRISPR/Cas9
genome editing. Our results highlight the importance of cytokinesis in the pathology of primary microcephaly.

Primary microcephaly is characterized by reduced head ations in mitotic-spindle regulation, account for approxi-
size accompanied by variable degrees of intellectual, lan- mately 40% of MCPH cases.1 Recently, a hierarchy of
guage, and motor-skill disability, but not progressive MCPH-associated proteins co-recruiting to the centrosome
cognitive decline, spasticity, or epilepsy.1 In autosomal- was described as mediating centrosomal duplication,5 sug-
recessive primary microcephaly (MCPH [MIM: 251200]), gesting a critical role for these MCPH-associated proteins
the cerebral cortex is particularly reduced in size, leading in centrosomal biogenesis.
to an apparently simplified gyral pattern because Because centrosomes play multiple roles in the cell (e.g.,
mantle thickness is grossly preserved but surface area is serving as microtubule-organizing centers, as spindle poles
dramatically reduced.2 MRI of an affected fetus with a during mitosis, and as basal bodies in ciliogenesis), the
prenatal diagnosis has shown the frontal lobes of the cellular etiology of MCPH has remained unclear. Initial
cerebral cortex to be affected as early as 30 weeks of studies focused on the roles of MCPH-associated proteins
gestation.3 at the spindle pole in determining mitotic-spindle orienta-
MCPH is considered a disorder of neural progenitor cell tion. The position of the centrosome in the dividing cell
(NPC) proliferation or survival during embryogenesis, pre- regulates mitotic orientation, which can, in turn, regulate
dominantly of autosomal-recessive or X-linked inheri- neural stem cell fate decisions.6 Indeed, defects in the
tance. Mutations in 16 loci (MCPH1MCPH16) have orientation of the mitotic cleavage plane contribute to
been described as associated with MCPH, and many of neurogenesis defects in an MCPH model.7 Recent work
these loci encode proteins implicated in the biogenesis has extended the roles of centrosomes in microcephaly
and function of the centrosome, an organelle critical for to include a potential function in cilia. MCPH-associated
multiple cellular functions.4 MCPH-associated genes proteins play essential roles in cilia, which are critical for
encode essential centrosomal-duplication proteinsSAS- cells to process Sonic hedgehog signals that can regulate
6, STIL, CPAP, CEP63, CEP135, and CEP152and pericen- neurogenesis.8 Furthermore, cells harboring mutations in
triolar-matrix proteinsWDR62, ASPM, and CEP215 (also microcephaly-associated genes can show disrupted cilio-
known as CDK5Rap2). Most of these mutations generate genesis,9,10 but classically, ciliopathy disorders do not
premature stop codons that predominantly result in demonstrate microcephaly. Thus, identifying non-centro-
nonsense-mediated mRNA decay and absent proteins. somal factors that lead to MCPH when mutated could
ASPM (MIM: 605481) mutations, which result in alter- help clarify mechanisms.

1
Howard Hughes Medical Institute, Rady Childrens Institute of Genomic Medicine, University of California, San Diego, San Diego, CA 92093, USA; 2Lab-
oratory for Pediatric Brain Disease, The Rockefeller University, New York, NY 10065, USA; 3Department of Human Genetics, School of Medicine, University
of Michigan, Ann Arbor, 48109 MI, USA; 4Clinical Genetics Department, Human Genetics and Genome Research Division, National Research Centre, Cairo
12311, Egypt; 5Division of Cardiology, Department of Medicine, University of California, San Diego, San Diego, CA 92093, USA; 6Broad Institute of MIT and
Harvard, Cambridge, MA 02141, USA; 7Yale Program on Neurogenetics, Departments of Neurosurgery, Neurobiology, and Genetics, School of Medicine,
Yale University, New Haven, CT 06510, USA; 8Department of Medical Genetics, School of Medicine, Istanbul Bilim University, Istanbul 34394, Turkey;
9
Department of Pediatrics, Istanbul Bilim University, Istanbul 34394, Turkey
*Correspondence: [email protected] (M.G.), [email protected] (J.G.G.)
http://dx.doi.org/10.1016/j.ajhg.2016.07.004.
2016 American Society of Human Genetics.

The American Journal of Human Genetics 99, 110, August 4, 2016 1

 Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

To identify additional mechanisms of microcephaly, we missense variant (c.376A>C [p. Lys126Gln]; hg19 chr12:
recruited individuals displaying MCPH in the setting of g.120295365T>G) also affecting the kinase domain. We
parental consanguinity. Subjects were enrolled according noticed that subject 1379-B1-1 was more severely impaired
to protocols approved by institutional review boards than other affected individuals from this family. Whether
at affiliated institutions. We performed whole-exome other variants from this individual (Table S2) contribute
sequencing (WES) on at least one affected member per to his clinical symptoms requires further investigation.
family and focused on the identification of potentially Family 1924 from Turkey had a single male child affected
deleterious rare homozygous variants.11 Three indepen- with MCPH from a consanguineous marriage. He dis-
dent consanguineous families whose children showed played a reduced HC of 45 cm (
6.5 SDs), intellectual
non-syndromic MCPH presented with homozygous disability, delayed motor and speech development, and
missense variants in CIT (MIM: 605629), encoding citron large protruding ears. WES detected a homozygous variant
rho-interacting kinase (CIT), which has established roles (c.689A>T [p.Asp230Val]; hg19 chr12: g.120270639T>A)
in cytokinesis. The variants were unique in our dataset of also affecting the encoded kinase domain. It is worth
more than 5,000 geographically matched individuals noting that another common feature of the MCPH-
sequenced by exome, were not represented in the Greater affected individuals from these three families is short stat-
Middle Eastern Variome or the Exome Aggregation Con- ure, which is also seen in individuals with MCPH1 muta-
sortium (ExAC) Browser, were fully segregated in the tions12 but is not a common finding in other MCPH cases.
families according to a recessive mode of inheritance CIT was identified as a 183 kDa GTP-bound Rac and Rho
(Figure S1A), and were predicted to be damaging with binding protein.13 There are two cDNA isoforms: (1) one
altered evolutionarily conserved amino acids (Figure S1B (GenBank: NM_001206999.1) encoding a 2,027 aa full-
and Table S1). Moreover, none of the families displayed length CIT-K, which has N-terminal kinase, coiled-coil,
functionally relevant variants in any of the previously es- zinc finger, Pleckstrin homology, and citron homology do-
tablished MCPH-associated genes in OMIM, and no other mains,14 and (2) another (GenBank: NM_007174.2) encod-
variants that met screening criteria showed segregation ac- ing a 1,545 aa CIT-N, which is produced by alternative
cording to a recessive fully penetrant mode of inheritance transcriptional initiation and lacks the kinase domain
(Table S1). but maintains the other C-terminal domains (Figures 1C
Family 718 from Egypt had four children affected by and 1D).15 CIT-K mRNA is specifically expressed in prolifer-
MCPH from a consanguineous marriage (Figure 1A and ating neural precursors in the developing brain, whereas
Table 1). Individual 718-IV-1 was born at full term with a CIT-N mRNA is expressed by post-mitotic cells,16 suggest-
reduced head circumference (HC) of 32.5 cm (
1 SD) ing a role for the kinase in neurogenesis. CIT-K localizes
and an average body weight. Developmental delay with to the cleavage furrow and midbody, where it promotes
motor, speech, and social impairment was noted at 3 years. cytokinesis during cell division.17
At 9 years, HC was 44.8 cm (
5.6 SDs). He displayed The CIT variants that we identified were notable for
a sloping forehead (Figure 1B), moderate intellectual several reasons. First, variants were not observed in the
disability, mild hypertonia, and brisk reflexes. His sister ExAC Browser or in our collective ethnically matched
(718-IV-2) had a similar clinical course and had a HC of database of 10,000 WES traces from families affected by
43 cm (
7.4 SDs) at 8 years of age. Similarly, 718-IV-3 neurodevelopmental disease. Second, all variants occurred
was noted to have microcephaly at birth but died on the within the kinase domain of the protein. Third, all variants
first day of life as a result of unrelated causes, and 718-IV- altered highly conserved residues, not just in evolution-
4 had a HC of 41 cm (
5.6 SDs) at age 2 years and mild arily orthologs of CIT but also in other sequence-similar
intellectual disability. Clinical features were not progres- kinase family members, including ROCK1 and AKT1
sive, and no spasticity or epileptic seizures were described. (Figure S1C). In fact, p.Lys126Gln, the amino acid substitu-
Brain MRI of 718-IV-1, 718-IV-2, and 718-IV-4 revealed a tion detected in family 1379, was in the invariant lysine
simplified gyral pattern and a thin corpus callosum (K) residue in subdomain II, which is present in all known
(Figure 1B). WES identified a homozygous missense variant kinases and is involved in the phosphotransfer reaction.18
(c.317G>T [p.Gly106Val]; hg19 chr12: g.120295424C>A; Fourth, using the resolved structure of the ROCK1 kinase
GenBank: NM_001206999.1) affecting the kinase domain domain to predict the function of the altered amino acids
of CIT (Figures 1C and 1D). in CIT,19 we found that two variants (p.Gly106Val and
Family 1379 from Egypt had two first-cousin parental p.Lys126Gln) were within the ATP binding pocket of the
consanguineous branches and three children affected by kinase domain, and one (p.Asp230Val) was within the
MCPH. All exhibited reduced head size, intellectual DFG (Asp-Phe-Gly) motif, which positions ATP for phos-
disability, delayed motor and speech development, phoryl transfer20 (Figure S1D). These findings suggest
hypertonia, brisk reflexes, sloping foreheads, and relatively that the mutations impair kinase activity.
large ears. HC was
7 to
8.4 SDs in the first decade of To test for impaired kinase activity, we FLAG tagged
life. MRI of individual 1379-IV-B1-1 demonstrated a amino acids 1480 of CIT-K, encoding the kinase domain,
simplified gyral pattern and a thin corpus callosum, similar either as wild-type (WT) or with each identified CIT variant
to those in family 718. WES revealed a homozygous engineered. The tagged proteins were immunoprecipitated

2 The American Journal of Human Genetics 99, 110, August 4, 2016

 Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

Figure 1. Mutations in CIT Cause Primary Microcephaly

(A) Pedigrees of consanguineous families 718, 1379, and 1924. Symbols are as follows: filled, affected; empty, unaffected; circle, female;
square, male; hash, deceased; B, branch.
(B) Faces (top) and axial MRI (bottom) of representative affected individuals from each family show reduced brain volume and a simpli-
fied gyral pattern, consistent with a diagnosis of MCPH.
(C) Exonic structure of CIT and the location of the identified mutations. The shorter CIT-N isoform, encoded by GenBank: NM_007174.
2, lacks the N-terminal kinase domain. The longer CIT-K isoform, encoded by GenBank: NM_001206999.1, encodes the kinase domain
where the CIT variants localize.
(D) Identified missense variants cluster within the kinase domain of full length CIT-K.
(E) Defective activity of the kinase domain with CIT variants. P[32] incorporation was detected in the wild-type only for histone H1 and
FLAG-CIT autophosphorylation.

from transfected 293T cells with anti-FLAG antibody and during cell division. During cytokinesis, the contractile
then used in an in vitro kinase assay. We utilized a ki- ring forms beneath the cell equatorial surface to form the
nase-dead (p.Lys126Ala) version with an alanine substitu- cleavage furrow, and then ingression of the furrow results
tion at the ATP donor site as a negative control.17 As in the formation of an intercellular bridge called the mid-
reported, WT CIT-K exhibited kinase activity toward exog- body. The cell cycle completes as the midbody is resolved
enous histone H1 substrate, as well as autophosphoryla- and the two daughter cells separate, a process known as
tion activity.21 Neither of these kinase activities was abscission.22 Disrupted cytokinesis frequently leads to
observed from the clone harboring p.Lys126Ala. Further- binucleated cells, aneuploidy, chromosomal instability,
more, none of the clones with CIT variants showed phos- activation of p53, cell-cycle arrest, and apoptosis. CIT-K is
phorylation of histone H1 or autophosphorylation on localized in the midbody during abscission, and knock-
the basis of P[32] incorporation (Figure 1E), suggesting down can result in failed abscission followed by re-fusion
that all three variants impair kinase activity. of the two daughter cells.23
Cytokinesis refers to the final stage of the cell cycle, in To study the functional consequences of the CIT variants
which the physical separation of two daughter cells occurs at the cellular level, we collected skin-derived fibroblasts

The American Journal of Human Genetics 99, 110, August 4, 2016 3

 4

Table 1. Clinical Features of the Affected Individuals in This Study

The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics 99, 110, August 4, 2016

718-IV-1 718-IV-2 718-IV-4 1379-IV-B2-1 1379-IV-B2-3 1379-IV-B1-1 1924-IV-1

Gender male female male female female male male

Ethnic origin Egypt Egypt Egypt Egypt Egypt Egypt Turkey

Pregnancy duration term term term term term term term

Weight at birth 3.2 kg (
0.2 SD) 3 kg (
0.4 SD) 3.5 kg (0.1 SD) 2.5 kg (
1.9 SDs) 2.5 kg (
1.9 SDs) 2 kg (
2.4 SDs) NA

Length at birth 48 cm (
0.4 SD) 49 cm 50 cm (0.4 SD) 50 cm (0.4 SD) 49 cm 49.5 cm (0.2 SD) NA

HC at birth 31 cm 31.5 cm 32 cm 31 cm 30 cm 30 cm NA

HC at last examination 44.8 cm (
5.6 SDs) 43 cm (
7.4 SDs) 41 cm (
5.6 SDs) 41.2 cm (
7.8 SDs) 40 cm (
8.4 SDs) 39.5 cm (
8.3 SDs) 45 cm (
6.5 SDs)

Diagnosis age 3 years 2 years at birth 5 years 1 year 1 year 4 years

Intellectual disability moderate mild mild moderate moderate severe moderate

Development

Gross motor delayed delayed delayed delayed delayed delayed delayed

Fine motor delayed delayed normal delayed delayed absent delayed

Language delayed delayed delayed delayed delayed absent delayed

Social delayed delayed delayed delayed delayed delayed delayed

Seizures

Present

Neurological Findings

Hypertonia mild mild
mild mild severe, acquired

arthrogryposis

Hypotonia

Deep tendon reflexes brisk brisk normal brisk brisk brisk normal

Spastic tetraplegia

Ataxia

Investigations

Metabolic normal normal NA NA NA normal normal

VEP and ERG normal normal normal NA NA normal normal

EEG normal normal normal NA NA generalized normal

epileptogenic activity

MRI

Simplified gyral NA NA
pattern

(Continued on next page)

Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

from affected and carrier individuals from families 718 and

1379, from affected individuals from family 1924, and

lips, large ears

from a healthy unrelated control individual. Primary fibro-

hairline, full
low anterior
1924-IV-1 blasts were unremarkable in culture and displayed no de-
fects in cell proliferation or mitosis (data not shown).

NA

NA

NA
Therefore, we generated induced pluripotent stem cells

(iPSCs) through reprograming by integration-free episomal
methods.24 We excluded gross chromosomal post-reprog-

(R), large ears, retruded

mandible, full lips and
flat occiput, esotropia

cheeks, tubular nose

ramming rearrangements. Furthermore, we found no

104 cm (
0.9 SD)

defect in differentiation of iPSCs into mesodermal or endo-
1379-IV-B1-1

dermal lineages.
From iPSCs, we next generated NPCs by using a dual-
SMAD inhibition protocol.25 As expected, amounts of

mild

Abbreviations are as follows: EEG, electroencephalography; ERG, electroretinography; HC, head circumference; NA, not available; R, right; and VEP, visual evoked potential.

PAX6 and CIT-K in mutant NPCs were comparable to those
in related controls (Figures S2A and S2B). CIT-K was de-

and cheeks, large ears

tubular nose, full lips
tected and localized to the midbody core in a manner indis-
102 cm (
1.2 SDs)

sloping forehead,
tinguishable from that of the wild-type during cytokinesis
1379-IV-B2-3

(Figure S2C), suggesting that the absence of kinase activity

does not lead to protein mislocalization. Furthermore, mid-
body integrity was intact, as evidenced by indistinguishable
NA

NA

NA

NA

localization of Aurora B to the midbody flank.26

To assess for functional defects in cytokinesis, we used

and cheeks, large ears
tubular nose, full lips

time-lapse imaging to monitor cell-cycle progression by

124 cm (
1.5 SDs)

sloping forehead,

comparing the entire duration of mitosis in NPCs. Previous

1379-IV-B2-1

work in HeLa cells showed that knockdown of CIT resulted

in delayed cytokinesis and blebbing of the cellular mem-
brane, followed by either early fusion or late fusion of
NA

NA

NA

NA

the daughters with binucleated derivatives.23 In mutant

NPCs, we found a dramatic increase in the length of cyto-

and cheeks, large ears
tubular nose, full lips

kinesis and cellular blebbing in all mitotic cells recorded.

This resulted in one of two outcomes. In about 25%
sloping forehead,
86 cm (mean)

of cells, blebbing was followed by failed cytokinesis and

the formation of cellular fusions and binucleated cells
718-IV-4

(Figure 2A, middle). In the remainder, after the delay,

cytokinesis completed with separation of the two daughter

cells (Figure 2A, bottom). On average, the length of

cytokinesis was doubled in mutant cells (mean 5 SEM:
and cheeks, large ears
tubular nose, full lips
119 cm (
1.2 SDs)

73.6 5 4.1 min versus 33.5 5 1.4 min).

sloping forehead,

Failure to achieve cytokinesis can result in subsequent

multipolar spindles as a result of duplicated centrosomes
718-IV-2

and nuclear material. To evaluate for multipolar spindles,

we fixed and stained sub-confluent NPCs for a-tubulin

and DAPI and then counted the percentage of mitotic fig-

ures with three or more spindle poles. Multipolar spindles
and cheeks, large ears
tubular nose, full lips

were evident in about 28% of mitotic cells, whereas they

sloping forehead,
125 cm (
1 SD)

were evident in fewer than 5% of control cells. We

conclude that multipolar spindles are a frequent feature
718-IV-1

of CIT-mutated NPCs.
To test whether the mutation caused the observed

cellular phenotypes, we corrected the c.317G>T

(p.Gly106Val) mutation by using CRISPR/Cas9-based
Hypogenesis of corpus
Continued

Cerebellar hypoplasia

Brainstem hypoplasia

genome editing in iPSCs derived from affected individual

718-IV-1 by homologous recombination (Figure 3A). A
Miscellaneous

Autistic features

Dysmorphism
Optic atrophy
abnormalities
White-matter

genome-editing donor plasmid containing wild-type CIT

Short stature

exon 4, flanking genomic sequence, and a puromycin

callosum
Table 1.

cassette (LoxP-Puro-LoxP) was electroporated together

with Cas9- and gRNA-expressing plasmids into iPSCs.

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 Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

Figure 2. Cell-Division Defects in NPCs Derived from Individuals with CIT Variants
(A) Representative phase-contrast time-lapse images of NPCs from unaffected and affected individuals. 718-III-1 is a healthy father (black
typeface), and 718-IV-1 is an affected member (red typeface). Asterisks mark the dividing cells analyzed in each series. Note that unaf-
fected cells completed division within 28 min. Affected cells (top row) showed blebbing at 16 min and then incomplete cytokinesis with
binucleated derivatives at 28 min. Affected cells (bottom row) showed minimal blebbing and then division by 48 min. Arrows mark
cortical blebbing.
(B) Quantification of the length of cytokinesis (n 15 cells per group).
(C and D) Representative images and quantification of the percentage of multipolar cells in NPCs from unaffected (C) and affected (D)
individuals. Arrowheads mark multipolar cells (n 7 cultures per group).
Bar graphs show the mean 5 SEM. **p < 0.01 (Students t test). Scale bar represents 10 mm.

Individual iPSC clones were propagated after puromycin corrected NPCs (Figures 3D and 3E). We conclude that
selection. After successful biallelic targeting (Figures S3A the CIT variants identified from the affected individual
S3C), the puromycin cassette was removed by transfection were necessary for mediating defects in cytokinesis, given
with a plasmid expressing Cre recombinase (Figure S3D). that cellular defects were corrected when the wild-type
Of note, the expression level of CIT was indistinguishable allele was genetically engineered into mutant cells.
between affected and wild-type cells after puromycin Both prolonged and incomplete cell division are associ-
recombination in corrected lines (data not shown), sug- ated with genotoxic stress and increased apoptotic cell
gesting that genome editing did not adversely affect death22,27 and are proposed as mechanisms in mouse
expression. We then differentiated cells into NPCs and per- and zebrafish MCPH models.2830 Furthermore, Cit-defi-
formed time-lapse microscopy to evaluate cytokinesis. cient mice display binucleated cells and increased
Corrected cells showed no delay in the length of cytoki- apoptosis in the developing cerebral cortex.16 To test for
nesis (Figures 3B and 3C). Furthermore, there was no apoptosis in a complex cellular milieu, we generated neu-
accumulation above baseline of multiple spindle cells in rospheres from control and mutant cells, together with

6 The American Journal of Human Genetics 99, 110, August 4, 2016

 Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

Figure 3. Correcting the CIT Variant Rescues Cellular NPC Phenotypes in Cells from Affected Individuals
(A) Schematic of gene-editing strategy for correcting the CIT variant in iPSCs. WT exon 4 and the floxed puromycin cassette were recom-
bined at the mutant locus after Cas9 cleavage. Cre-mediated removal of the LoxP-Puro-LoxP cassette left only the single LoxP site.
(legend continued on next page)

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 Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

CIT-mutation-corrected cells, and then stained for cleaved Our data align with those of other studies arguing
caspase-3 as a marker of apoptosis. Dramatic evidence for against MCPH as a primary defect of centrosomes for the
increased apoptosis (an overall 4-fold increase in intensity) following reasons. First, fruit flies without centrosomes still
was detected in mutant but not control or mutation-cor- undergo neurogenesis,38 suggesting that centrosomes are
rected neurospheres (Figures 3F and 3G). not required for neuronal proliferation. Second, mutations
Here, we describe families affected by recessive MCPH in microcephaly-associated genes Sas4 and Cep63 in mice
caused by biallelic missense CIT alleles affecting the kinase lead to genotoxic stress, activation of the p53 pathway,
domain and thus leading to undetectable kinase activity. and apoptosis, probably independent of cleavage plane ef-
These mutations were remarkable in that they did not alter fects.39 Third, neurogenesis defects in microcephaly mouse
the expression level of the mRNA or CIT localization in models have been rescued at least in part by concurrent
anaphase, suggesting that the kinase activity of CIT is removal of p53.39,40 The data support a model in at least
required for function in controlling cytokinesis. Previous some forms of microcephaly, whereby loss of mitotic integ-
studies using overexpression of kinase-dead CIT in HeLa rity through an effect on centrosomes or cytokinesis can
cells found largely retained effects, but these studies could result in genome instability, genotoxic stress, apoptosis,
not differentiate between loss-of-function and toxic gain- and subsequently, reduced cerebral volume.
of-function effects.17 The fact that the parent and sibling
carriers were healthy and had a normal HC suggests that
Accession Numbers
our mutations did not act in a dominant-negative fashion,
and because no individuals with truncating CIT mutations The WES data for all study subjects who consented to data release
were detected with MCPH, it is tempting to speculate that have been deposited under accession number dbGaP: phs000288.
CIT retains some biological activity even in the setting of
kinase loss. This is further supported by the finding of Supplemental Data
the more severe microlissencephaly phenotype observed
Supplemental Data include three figures and two tables and can be
among individuals with truncating CIT mutations, shown
found with this article online at http://dx.doi.org/10.1016/j.ajhg.
in the accompanying paper by Li et al.31
2016.07.004.
CIT is classified as an AGC kinase (by homology with
protein kinases A, G, and C), which are regulated by second
messengers, such as cyclic AMP (PKA) and lipids (PKC).32 Acknowledgments
CIT binds activated Rho and Rac,13 and activated Rho We thank Joseph LoTurco and Ferdinando Di Cunto for communi-
correlates with the translocation of CIT to the cleavage cating unpublished results and Susan Taylor for providing sug-
furrow.33 Most AGC kinases require sequential phosphory- gestions on the project. We thank the subjects and their families
lations of the turn and hydrophobic motifs to stabilize for their contributions to this study. This work was supported
the active conformation,34 but it is still unclear whether by the NIH (R01NS041537, R01NS048453, R01NS052455, and
other kinases regulate CITs kinase activity. Although P01HD070494 to N.C.), the Howard Hughes Medical Institute
CIT can function with proteins such as KIF14 and (J.G.G.), and the Druckenmiller Fellowship from the New York
TUBB3,26,35 direct phosphorylation targets of CIT have Stem Cell Foundation (to H.L). We thank the Broad Institute
not yet been identified. (U54HG003067 to E. Lander) and the Yale Center for Mendelian
Disorders (U54HG006504 to R. Lifton) for sequencing support.
Several spindle-pole-localized MCPH-associated proteins,
including ASPM, CENPJ, and CDK5RRAP2, are also present
Received: May 1, 2016
in the midbody during cytokinesis.36 Moreover, loss of Accepted: July 5, 2016
ASPM in cultured cells leads to cytokinesis failure followed Published: July 21, 2016
by apoptosis, in addition to misorientation of the mitotic
spindle.37 Our results confirm that cytokinesis failure and
subsequent apoptosis are underlying mechanisms for the Web Resources
genetic forms of MCPH. Interestingly, the C terminus of dbGaP, http://www.ncbi.nlm.nih.gov/gap
ASPM interacts with CIT-K, suggesting that CIT-K and Exome Aggregation Consortium (ExAC) Browser, http://exac.
ASPM might function together in regulating cytokinesis.36 broadinstitute.org/

(B) Representative differential interference contrast (DIC) time-lapse images from unaffected (black), affected (red), and mutation-cor-
rected (gray) NPCs during cytokinesis. Scale bar represents 10 mm.
(C) Quantification of the length of cytokinesis (n 15 cells for each group).
(D and E) Representative images and quantification of the percentage of multipolar cells in unaffected, affected, and mutation-corrected
(COR) NPCs. Arrowheads mark multipolar cells (n 4 cultures per group). Scale bar represents 10 mm.
(F and G) Representative images (F) and quantification (G) of active caspase-3 immunoreactivity in unaffected, affected, and mutation-
corrected neurospheres (n 6 cultures per group). Note the dramatically increased amount of cleaved caspase-3 in mutant cells (718-
IV-1) prior to correction (F, arrow). Scale bar represents 150 mm.
(C, E, and G) One-way ANOVA was followed by Tukeys multiple-comparison test.
Bar graphs show the mean 5 SEM. **p < 0.01.

8 The American Journal of Human Genetics 99, 110, August 4, 2016

 Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

Greater Middle East Variome, http://igm.ucsd.edu/gme 12. Neitzel, H., Neumann, L.M., Schindler, D., Wirges, A., Tonnies,
NHLBI Exome Sequencing Project (ESP) Exome Variant Server, H., Trimborn, M., Krebsova, A., Richter, R., and Sperling, K.
http://evs.gs.washington.edu/EVS/ (2002). Premature chromosome condensation in humans asso-
OMIM, http://www.omim.org/ ciated with microcephaly and mental retardation: a novel auto-
PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/ somal recessive condition. Am. J. Hum. Genet. 70, 10151022.
PROVEAN, http://provean.jcvi.org/ 13. Madaule, P., Furuyashiki, T., Reid, T., Ishizaki, T., Watanabe,
RefSeq, http://www.ncbi.nlm.nih.gov/refseq/ G., Morii, N., and Narumiya, S. (1995). A novel partner for
SeattleSeq, http://snp.gs.washington.edu/SeattleSeqAnnotation137/ the GTP-bound forms of rho and rac. FEBS Lett. 377, 243248.
SIFT, http://sift.jcvi.org/ 14. Watanabe, S., De Zan, T., Ishizaki, T., and Narumiya, S. (2013).
Citron kinase mediates transition from constriction to abscis-
sion through its coiled-coil domain. J. Cell Sci. 126, 1773
1784.
References 15. Camera, P., da Silva, J.S., Griffiths, G., Giuffrida, M.G., Ferrara,
1. Faheem, M., Naseer, M.I., Rasool, M., Chaudhary, A.G., Kumo- L., Schubert, V., Imarisio, S., Silengo, L., Dotti, C.G., and Di
sani, T.A., Ilyas, A.M., Pushparaj, P., Ahmed, F., Algahtani, Cunto, F. (2003). Citron-N is a neuronal Rho-associated pro-
H.A., Al-Qahtani, M.H., and Saleh Jamal, H. (2015). Molecular tein involved in Golgi organization through actin cytoskel-
genetics of human primary microcephaly: an overview. BMC eton regulation. Nat. Cell Biol. 5, 10711078.
Med. Genomics 8 (Suppl 1 ), S4. 16. Di Cunto, F., Imarisio, S., Hirsch, E., Broccoli, V., Bulfone, A.,
2. Adachi, Y., Poduri, A., Kawaguch, A., Yoon, G., Salih, M.A., Ya- Migheli, A., Atzori, C., Turco, E., Triolo, R., Dotto, G.P., et al.
mashita, F., Walsh, C.A., and Barkovich, A.J. (2011). Congen- (2000). Defective neurogenesis in citron kinase knockout
ital microcephaly with a simplified gyral pattern: associated mice by altered cytokinesis and massive apoptosis. Neuron
findings and their significance. AJNR Am. J. Neuroradiol. 32, 28, 115127.
11231129. 17. Madaule, P., Eda, M., Watanabe, N., Fujisawa, K., Matsuoka, T.,
3. Desir, J., Cassart, M., David, P., Van Bogaert, P., and Abramo- Bito, H., Ishizaki, T., and Narumiya, S. (1998). Role of citron
wicz, M. (2008). Primary microcephaly with ASPM mutation kinase as a target of the small GTPase Rho in cytokinesis.
shows simplified cortical gyration with antero-posterior Nature 394, 491494.
gradient pre- and post-natally. Am. J. Med. Genet. A. 146A, 18. Carrera, A.C., Alexandrov, K., and Roberts, T.M. (1993). The
14391443. conserved lysine of the catalytic domain of protein kinases
4. Woods, C.G., and Basto, R. (2014). Microcephaly. Curr. Biol. is actively involved in the phosphotransfer reaction and not
24, R1109R1111. required for anchoring ATP. Proc. Natl. Acad. Sci. USA 90,
5. Kodani, A., Yu, T.W., Johnson, J.R., Jayaraman, D., Johnson, 442446.
T.L., Al-Gazali, L., Sztriha, L., Partlow, J.N., Kim, H., Krup, 19. Jacobs, M., Hayakawa, K., Swenson, L., Bellon, S., Fleming, M.,
A.L., et al. (2015). Centriolar satellites assemble centrosomal Taslimi, P., and Doran, J. (2006). The structure of dimeric
microcephaly proteins to recruit CDK2 and promote centriole ROCK I reveals the mechanism for ligand selectivity. J. Biol.
duplication. eLife 4, 4. Chem. 281, 260268.
6. Kaltschmidt, J.A., Davidson, C.M., Brown, N.H., and Brand, 20. Kornev, A.P., Haste, N.M., Taylor, S.S., and Eyck, L.F. (2006).
A.H. (2000). Rotation and asymmetry of the mitotic spindle Surface comparison of active and inactive protein kinases
direct asymmetric cell division in the developing central ner- identifies a conserved activation mechanism. Proc. Natl.
vous system. Nat. Cell Biol. 2, 712. Acad. Sci. USA 103, 1778317788.
7. Fish, J.L., Kosodo, Y., Enard, W., Paabo, S., and Huttner, W.B. 21. Di Cunto, F., Calautti, E., Hsiao, J., Ong, L., Topley, G., Turco,
(2006). Aspm specifically maintains symmetric proliferative E., and Dotto, G.P. (1998). Citron rho-interacting kinase, a
divisions of neuroepithelial cells. Proc. Natl. Acad. Sci. USA novel tissue-specific ser/thr kinase encompassing the Rho-
103, 1043810443. Rac-binding protein Citron. J. Biol. Chem. 273, 2970629711.
8. Tong, C.K., Han, Y.G., Shah, J.K., Obernier, K., Guinto, C.D., 22. Green, R.A., Paluch, E., and Oegema, K. (2012). Cytokinesis in
and Alvarez-Buylla, A. (2014). Primary cilia are required in a animal cells. Annu. Rev. Cell Dev. Biol. 28, 2958.
unique subpopulation of neural progenitors. Proc. Natl. 23. Gai, M., Camera, P., Dema, A., Bianchi, F., Berto, G., Scarpa, E.,
Acad. Sci. USA 111, 1243812443. Germena, G., and Di Cunto, F. (2011). Citron kinase controls
9. Waters, A.M., Asfahani, R., Carroll, P., Bicknell, L., Lescai, F., abscission through RhoA and anillin. Mol. Biol. Cell 22, 3768
Bright, A., Chanudet, E., Brooks, A., Christou-Savina, S., Os- 3778.
man, G., et al. (2015). The kinetochore protein, CENPF, is 24. Okita, K., Matsumura, Y., Sato, Y., Okada, A., Morizane, A.,
mutated in human ciliopathy and microcephaly phenotypes. Okamoto, S., Hong, H., Nakagawa, M., Tanabe, K., Tezuka,
J. Med. Genet. 52, 147156. K., et al. (2011). A more efficient method to generate integra-
10. Hu, W.F., Pomp, O., Ben-Omran, T., Kodani, A., Henke, K., Mo- tion-free human iPS cells. Nat. Methods 8, 409412.
chida, G.H., Yu, T.W., Woodworth, M.B., Bonnard, C., Raj, 25. Novarino, G., El-Fishawy, P., Kayserili, H., Meguid, N.A., Scott,
G.S., et al. (2014). Katanin p80 regulates human cortical devel- E.M., Schroth, J., Silhavy, J.L., Kara, M., Khalil, R.O., Ben-Om-
opment by limiting centriole and cilia number. Neuron 84, ran, T., et al. (2012). Mutations in BCKD-kinase lead to a
12401257. potentially treatable form of autism with epilepsy. Science
11. Dixon-Salazar, T.J., Silhavy, J.L., Udpa, N., Schroth, J., Bielas, 338, 394397.
S., Schaffer, A.E., Olvera, J., Bafna, V., Zaki, M.S., Abdel- 26. Gruneberg, U., Neef, R., Li, X., Chan, E.H., Chalamalasetty,
Salam, G.H., et al. (2012). Exome sequencing can improve R.B., Nigg, E.A., and Barr, F.A. (2006). KIF14 and citron kinase
diagnosis and alter patient management. Sci. Transl. Med. act together to promote efficient cytokinesis. J. Cell Biol. 172,
4, 138ra78. 363372.

The American Journal of Human Genetics 99, 110, August 4, 2016 9

 Please cite this article in press as: Li et al., Biallelic Mutations in Citron Kinase Link Mitotic Cytokinesis to Human Primary Microcephaly,
The American Journal of Human Genetics (2016), http://dx.doi.org/10.1016/j.ajhg.2016.07.004

27. Pilaz, L.J., McMahon, J.J., Miller, E.E., Lennox, A.L., Suzuki, A., 34. Hauge, C., Antal, T.L., Hirschberg, D., Doehn, U., Thorup, K.,
Salmon, E., and Silver, D.L. (2016). Prolonged mitosis of neu- Idrissova, L., Hansen, K., Jensen, O.N., Jrgensen, T.J., Biondi,
ral progenitors alters cell fate in the developing brain. Neuron R.M., and Frodin, M. (2007). Mechanism for activation of the
89, 8399. growth factor-activated AGC kinases by turn motif phosphor-
28. McIntyre, R.E., Lakshminarasimhan Chavali, P., Ismail, O., ylation. EMBO J. 26, 22512261.
Carragher, D.M., Sanchez-Andrade, G., Forment, J.V., Fu, B., 35. Sgro, F., Bianchi, F.T., Falcone, M., Pallavicini, G., Gai, M.,
Del Castillo Velasco-Herrera, M., Edwards, A., van der Wey- Chiotto, A.M., Berto, G.E., Turco, E., Chang, Y.J., Huttner,
den, L., et al.; Sanger Mouse Genetics Project (2012). Disrup- W.B., and Di Cunto, F. (2016). Tissue-specific control of
tion of mouse Cenpj, a regulator of centriole biogenesis, phe- midbody microtubule stability by Citron kinase through
nocopies Seckel syndrome. PLoS Genet. 8, e1003022. modulation of TUBB3 phosphorylation. Cell Death Differ.
29. Novorol, C., Burkhardt, J., Wood, K.J., Iqbal, A., Roque, C., 23, 801813.
Coutts, N., Almeida, A.D., He, J., Wilkinson, C.J., and Harris, 36. Paramasivam, M., Chang, Y.J., and LoTurco, J.J. (2007). ASPM
W.A. (2013). Microcephaly models in the developing zebrafish and citron kinase co-localize to the midbody ring during cyto-
retinal neuroepithelium point to an underlying defect in kinesis. Cell Cycle 6, 16051612.
metaphase progression. Open Biol. 3, 130065. 37. Higgins, J., Midgley, C., Bergh, A.M., Bell, S.M., Askham, J.M.,
30. Kaindl, A.M., Passemard, S., Kumar, P., Kraemer, N., Issa, L., Roberts, E., Binns, R.K., Sharif, S.M., Bennett, C., Glover,
Zwirner, A., Gerard, B., Verloes, A., Mani, S., and Gressens, P. D.M., et al. (2010). Human ASPM participates in spindle orga-
(2010). Many roads lead to primary autosomal recessive nisation, spindle orientation and cytokinesis. BMC Cell Biol.
microcephaly. Prog. Neurobiol. 90, 363383. 11, 85.
31. Harding, B.N., Moccia, A., Soukarieh, O., Tubeuf, H., Drunat, 38. Basto, R., Lau, J., Vinogradova, T., Gardiol, A., Woods, C.G.,
S., Chitty, L.S., Verloes, A., Gressens, P., El Ghouzzi, V., Joriot, Khodjakov, A., and Raff, J.W. (2006). Flies without centrioles.
S., et al. (2016). Mutations in Citron Kinase Cause Recessive Cell 125, 13751386.
Microlissencephaly with Multinucleated Neurons. Am. J. 39. Marjanovic, M., Sanchez-Huertas, C., Terre, B., Gomez, R.,
Hum. Genet. 99. Published online July 21, 2016. http://dx. Scheel, J.F., Pacheco, S., Knobel, P.A., Martnez-Marchal, A.,
doi.org/10.1016/j.ajhg.2016.07.003. Aivio, S., Palenzuela, L., et al. (2015). CEP63 deficiency pro-
32. Pearce, L.R., Komander, D., and Alessi, D.R. (2010). The nuts and motes p53-dependent microcephaly and reveals a role for
bolts of AGC protein kinases. Nat. Rev. Mol. Cell Biol. 11, 922. the centrosome in meiotic recombination. Nat. Commun.
33. Eda, M., Yonemura, S., Kato, T., Watanabe, N., Ishizaki, T., Ma- 6, 7676.
daule, P., and Narumiya, S. (2001). Rho-dependent transfer of 40. Insolera, R., Bazzi, H., Shao, W., Anderson, K.V., and Shi, S.H.
Citron-kinase to the cleavage furrow of dividing cells. J. Cell (2014). Cortical neurogenesis in the absence of centrioles.
Sci. 114, 32733284. Nat. Neurosci. 17, 15281535.

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