TUBA1A mutations cause wide spectrum
lissencephaly (smooth brain) and suggest that
multiple neuronal migration pathways converge
on alpha tubulins
Ravinesh A. Kumar1, Daniela T. Pilz4, Timothy D. Babatz1, Thomas D. Cushion5,
Kirsten Harvey6, Maya Topf7, Laura Yates8, Stephanie Robb9, Go ¨khan Uyanik10,
Gracia M.S. Mancini11, Mark I. Rees4,5, Robert J. Harvey6and William B. Dobyns1,2,3,∗
1Department of Human Genetics,2Department of Neurology and3Department of Pediatrics, University of Chicago,
Chicago, IL 60637, USA,4Institute of Medical Genetics, University Hospital of Wales, Heath Park, Cardiff CF14 4XW,
UK,5Institute of Life Science, School of Medicine, Swansea University, Singleton Park SA2 8PP, UK,6Department of
Pharmacology, The School of Pharmacy, London WC1N 1AX, UK,7Institute of Structural and Molecular Biology,
Crystallography, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK,8Institute of Human
Genetics, International Centre for Life, Newcastle upon Tyne, NE1 3BZ, UK,9The Dubowitz Neuromuscular Centre,
Great Ormond Street Hospital, London WC1N 3JN, UK,10Institute of Human Genetics, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany and11Department of Clinical Genetics, Erasmus University Medical Center,
PO Box 2040, 3000CA Rotterdam, The Netherlands
Received February 26, 2010; Revised and Accepted April 30, 2010
We previously showed that mutations in LIS1 and DCX account for ∼85% of patients with the classic form of
lissencephaly (LIS). Some rare forms of LIS are associated with a disproportionately small cerebellum, referred
to as lissencephaly with cerebellar hypoplasia (LCH). Tubulin alpha1A (TUBA1A), encoding a critical structural
subunit of microtubules, has recently been implicated in LIS. Here, we screen the largest cohort of unexplained
LIS patients examined to date to determine: (i) the frequency of TUBA1A mutations in patients with lissence-
of different TUBA1A mutations on microtubule function. We identified novel and recurrent TUBA1A mutations
in ∼1% of children with classic LIS and in ∼30% of children with LCH, making this the first major gene associ-
ated with the rare LCH phenotype. We also unexpectedly found a TUBA1A mutation in one child with agenesis
of the corpus callosum and cerebellar hypoplasia without LIS. Thus, our data demonstrate a wider spectrum of
phenotypes than previously reported and allow us to propose new recommendations for clinical testing. We
also provide cellular and structural data suggesting that LIS-associated mutations of TUBA1A operate via
diverse mechanisms that include disruption of binding sites for microtubule-associated proteins (MAPs).
Development of the human brain requires an exquisitely
orchestrated program of neuronal migration and positioning.
Lissencephaly (LIS, smooth brain) comprises a group of
severe brain malformations associated with deficient neuronal
migration that results in mental retardation, epilepsy and when
severe a shortened lifespan (1–3). In the most severe form, the
∗To whom correspondence should be addressed at: Department of Human Genetics, University of Chicago, 920 East 58th Street, CLSC 319, Chicago,
IL 60637, USA. Tel: +1 7738343597; Fax: +1 7738348470; Email: email@example.com
# The Author 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
Human Molecular Genetics, 2010, Vol. 19, No. 14
Advance Access published on May 12, 2010
surface of the cerebral hemispheres is completely smooth
(agyria), whereas less severe forms of LIS are characterized
by simplified folding patterns with abnormally broad gyri
(pachygyria). Classic LIS (agyria–pachygyria spectrum) is
typically characterized by a loosely organized and markedly
thickened four-layer cortex (compared with the normal six-
layered cortical architecture) and apparently normal cerebel-
lum, although some patients have mild vermis hypoplasia
(4). However, some rare forms of LIS are associated with a
disproportionately small cerebellum, referred to as LIS with
cerebellar hypoplasia (LCH). Several subtypes are known
including LCH group b associated with Reelin pathway
defects and another type characterized by a very poorly orga-
nized two-layer cortex (4,5). Classic LIS and LCH most often
occur as isolated malformations, but rarely occur as part of
LIS has a strong genetic basis. Several genes that underlie
LIS are directly or indirectly associated with modulation of
microtubules, which are cytoskeletal elements involved in
key cellular processes including mitosis, cytokinesis, vesicle
transport, and neuronal migration. Heterozygous or hemizy-
gous mutations in the microtubule-associated genes LIS1 on
17p13.3 and DCX on Xq22.3q23 account for ?85% of
classic LIS (2,3,6,7). Co-deletion of the nearby LIS1 and
YWHAE genes underlies Miller–Dieker syndrome (8–10),
which consists of severe classic LIS and characteristic facial
dysmorphism. Mutations in several other genes including
ARX, RELN and VLDLR have been identified in other forms
of LIS but collectively these account for only a small pro-
portion of patients with LIS.
The most recent gene implicated in LIS encodes tubulin
alpha1A (TUBA1A) and resides on chromosome 12q13.12
(11–14). TUBA1A is a critical structural subunit of microtu-
bules that is transiently expressed during neuronal develop-
ment (15). Here, we report screening of the largest cohort
of LIS patients examined to date to determine the frequency
of TUBA1A mutations in patients with LIS, the spectrum of
phenotypes associated with TUBA1A mutations, and the func-
tional consequences of different TUBA1A mutations on micro-
We identified novel and recurrent TUBA1A mutations in
?1% of children with classic LIS and surprisingly in ?30%
of children with LCH spanning a wide spectrum of severity.
We also detected a novel TUBA1A mutation in one child
with agenesis of the corpus callosum (ACC) and cerebellar
hypoplasia (CBLH) without LIS. We provide structural and
functional data suggesting that LIS-associated mutations of
TUBA1A operate via diverse mechanisms, perturbing tubulin
a–b dimerization and most commonly affecting binding
sites for microtubule-associated proteins (MAPs).
TUABA1A mutations identified in 7% of unexplained
classic LIS and 32% of LCH
We screened the complete TUBA1A coding region and flank-
ing 5′- and 3′-untranslated regions using bi-directional
Sanger sequencing in 125 patients with LIS, including 72
patients with classic LIS, 22 with subcortical band heterotopia
(SBH), 29 with LCH and two with LIS and ACC. Mutations of
DCX and LIS1 were excluded by sequencing or by review of
brain imaging, so that patients with posterior-predominant
LIS (the LIS1 pattern) had normal sequencing results for
LIS1, but may or may not have had DCX testing, and vice
versa. We identified 5 missense mutations in 72 patients
with classic LIS (?7%) and 10 missense mutations in 29
patients with LCH (?34%) (Table 1). We subsequently
added mutations in two further patients with TUBA1A
mutations found by clinical testing (p.M377V and p.R390C),
but did not include them in our cohort analysis. Eight
mutations (p.L92V, p.V137D, p.D218Y, p.A270T, p.N329S,
p.M377V, p.R390C and p.M425K) were novel, while three
(p.R402C, p.R402H and p.R422H) were recurrent mutations
previously reported in other patients. We identified p.R402C
in five patients, p.R402H in two patients, and p.R422H in
Defining the phenotypic spectrum of TUBA1A mutations
Detailed brain imaging studies were available for all 17
patients with TUBA1A mutations. We immediately recognized
that mutations involving the arginine residue at codon 402
resulted in phenotypes indistinguishable from those associated
with mutations of the LIS1 gene, while mutations at other
codons produced more complex forms of LIS. From our
detailed review of these imaging studies, we were able to
delineate five distinct phenotype groups that are most easily
appreciated with multiple views in several planes, as shown
in Figs 1–4 and Supplementary Material, Figure S1.
The first group consists of five patients with the recurrent
p.R402C mutation (Fig. 1). All had classic LIS with frontal
pachygyria and posterior agyria corresponding to classic LIS
grade 3 with posterior more severe than anterior gradient,
rounded hippocampi with a thick outer rim (Ammon’s horn),
intact but dysmorphic corpus callosum (missing rostrum plus
flat genu and anterior body) and most often normal cerebellar
structure. We observed a slightly more severe gyral malfor-
mation intermediate between grades 2 and 3, and mild cerebel-
lar vermis hypoplasia in one patient each. This appearance is
identical to the most common pattern found with mutations
or deletions of the LIS1 gene.
Our second group consists of two patients with the recurrent
p.R402H mutation (Fig. 2). Both had severe, classic LIS with
nearly complete agyria, the same hippocampal and callosal
defects as in the first group and moderate cerebellar vermis
hypoplasia. This pattern resembles the brain imaging appear-
ance found in patients with the Miller–Dieker syndrome
(MDS) and co-deletion of LIS1 and YWHAE, except the cer-
ebellar vermis hypoplasia is more severe in our two patients.
The third group consists of five patients with mild or mod-
erate LCH and heterogeneous missense mutations (p.V137D,
p.A270T, p.M377V, p.R422H) throughout the gene (Fig. 3).
The brain imaging appearance is novel, consisting of diffuse
pachgyria with no areas of agyria, mild-to-moderate asymme-
try of the gyral malformation that could appear most severe
over either the central convexity or the posterior pole and a
thick cortex that is, however, not as thick as seen in patients
with LIS1 (or TUBA1A p.R402C or p.R402H) mutations.
The asymmetry is unusual for pachygyria; we considered
2818 Human Molecular Genetics, 2010, Vol. 19, No. 14
whether this could be polymicrogyria, but the cortex is too
thick and cortical infolding was not seen so we think this is
a variant of pachgyria and not polymicrogyria. The posterior
frontal, perisylvian and parietal regions were more severely
affected than the occipital poles, another striking difference
from the LIS1 mutation pattern in which the entire posterior
brain including the occipital poles is most severely involved.
One patient had a prominent deep cellular layer in the subcor-
tical perisylvian region that resembles a short band hetero-
topia, but the overlying cortex is not normal indicating
pachgyria and not SBH (Fig. 3N). The hippocampus is mal-
formed as in the previous groups. Our three patients with
severe pachygyria also had absent or nearly absent corpus cal-
losum, thin brainstem and severe cerebellar hypoplasia. The
cerebellar malformation consisted of small and upwardly
rotated vermis, cystic dilatation of the 4th ventricle, less
severe cerebellar hemisphere hypoplasia and mild to moder-
ately enlarged posterior fossa, thus meeting the criteria for
Dandy–Walker malformation, although the shape of the
vermis differed from the typical form of this disorder. The
least severely affected subjects had only moderate pachgyryia,
mildly dysmorphic corpus callosum with absent spenium and
mild cerebellar vermis hypoplasia.
Our fourth group consists of four patients with novel hetero-
zygous missense mutations associated with severe LCH that
corresponds to our previous LCH groups c, d and f (5) by
brain imaging (p.D218Y, p.N329S; p.M425K; Fig. 4) or fetal
neuropathology at 21 weeks gestation (p.L92V). In all, the
walls of the cerebral hemispheres are thin, and the cerebral
surface completely smooth except for a few abortive sulci at
the frontal pole in the third patient. The cortex demonstrates a
slightly different pattern ineach patient.Inthe first, the cerebral
wall is very thin, but the cortex and white matter can be distin-
guished (LR07-213; p.D218Y; Fig. 4A–E). In the second, the
cortex fills the entire mantle and creates an undulating pattern
along the walls of the lateral ventricles, a rare LIS pattern
(LR05-388; p.N329S; Fig. 4F–J). The same pattern was seen
in an LCH patient with no sample available to test (patient
LP99-059 shown in Fig. 6D–F in 5). In the third patient, the
cortex and white matter can barely be distinguished. The
amygdala and hippocampus are seen as small thickenings of
the temporal wall (LR08-388, p.M425K; Fig. 4K–O). The
corpus callosum is absent in all three patients. In the first two,
the brainstem is remarkably thin and the cerebellum consists
of a nubbin with the vermis ,1 cm in size. The posterior
fossa is mildly enlarged, but the overall appearance is atypical
for DWM because of the minute cerebella. In the third
patient, the brainstem is moderately small, and the cerebellar
defects meet the criteria for mild DWM.
The remaining subject was identified following fetal termin-
ation for ventriculomegaly and other anomalies at ?21 weeks
gestation (CM–66, p.L92V). The brain was small (weight
31 g, expected 45 g) and had a generalized deficiency of neur-
onal migration with neurons seen diffusely through the mantle
(thus no layers), a small amorphous group of neurons in the
area of the hippocampus, severe dilatation of the third and
lateral ventricles and absent corpus callosum. The brainstem
was very small with absent descending corticospinal tracts
and inferior olives and small basis pontis. The cerebellum
was also very small with absent dentate nuclei and formation
of only an indistinct cerebellar cortex.
Our last group consists of a single patient carrying a novel
de novo heterozygous missense mutation p.R390C with
mildly reduced number of gyri and shallow sulcal depth that
we designate as a ‘simplified’ gyral pattern, plus complete
ACC and moderate CBLH (Supplementary Material, Fig. S1).
The cortical-white matter interface appeared mildly irregular
in places, but the cortex was not thick and we found this uncon-
vincing for pachygyria, polymicrogyria or another cortical dys-
plasia; however, the resolution of the study was low. This
Cellular studies of TUBA1A mutations
Since all TUBA1A mutations reported to date are de novo and
affect a single allele of an autosomal gene, a dominant-
negative mechanism of action is plausible. Consistent with
this hypothesis, TUBA1A mutations reported to date do not
profoundly affect the ability of mutant TUBA1A to incorpor-
ate into the cytoskeleton in recombinant systems (12,16).
Table 1. TUBA1A mutations identified in LIS
Subject ID DNA IDDiagnosis StatusAmino acid Inheritancea
L92V (c.274C . G)
D218Y (c.652G . T)
A270T (c.808G . A)
N329S (c.986A . G)
R402C (c.1204C . T)
R402C (c.1204C . T)
R402C (c.1204C . T)
R402C (c.1204C . T)
R402C (c.1204C . T)
R402H (c.1205G . A)
R402H (c.1205G . A)
R422H (c.1265G . A)
R422H (c.1265G . A)
LCH×Sev group 4
LCH×mild group 3
LCH×Sev group 4
LCH×Mod group 3
LCH×Sev group 4
LIS×Mod group 1
LIS×Mod group 1
LIS×Mod group 1
LIS×Mod group 1
LIS×Mod group 1
LIS×Sev group 2
LIS×Sev group 2
LCH×Mod group 3
LCH×Mod group 3
LCH×Sev group 4
Sev, severe; Mod, moderate.
aParental DNA unavailable for testing.
Human Molecular Genetics, 2010, Vol. 19, No. 142819
To confirm this finding for a range of TUBA1A mutants
reported here and elsewhere, we introduced the mutations
I188L, P263T, R264C, L286F, R402H, R402C and S419L
into a construct expressing C-terminally FLAG-tagged
TUBA1A and transfected these into P19 cells (Fig. 5). Confo-
cal microscopy revealed that in each case, recombinant
TUBA1A is clearly made and the typical tubulin architecture
is observed. This suggests that these mutants readily incorpor-
ate into the cellular cytoskeletal network. This finding does not
support a recent study that reported haploinsufficiency as a
cause of the disease phenotype (16) and we chose to undertake
detailed structural modeling to predict the functional conse-
quences of existing and novel TUBA1A mutations.
Structural modeling of TUBA1A mutations
The N329S mutation is located on a-helix H10 of a-tubulin
(residues 324–337) (17) at the interface between a- and
b-tubulin monomers. H10 interacts with a-helix H6 (residues
206–214) and the adjacent loop (the B5–H5 loop, residues
171–183) on b-tubulin, both of which directly interact with
GTP (Fig. 6). In the wild-type structure, the interaction of
H10 with b-tubulin is stabilized by the side chains of two resi-
dues, K326 and N329. The K326 amino group forms an H
bond with the backbone oxygen of Y210 and is also involved
in a cation–p interaction with the aromatic ring of F214. The
N329 carboxamide group forms two H bonds, one with the
D179 carboxylate group and the other with the V177 backbone
oxygen (on the B5–H5 loop). The N329S mutation will not
allow the latter H bond interactions and therefore is likely to
destabilize the interaction between a- and b-tubulin mono-
mers around H6 and the B5–H5 loop, possibly affecting the
conformation necessary for GTP binding.
Six additional TUBA1A mutations cluster around a-helices
H11 and H12 of a-tubulin (R402H, R402C, S419L, R422H,
R422C and M425K) and they are likely to affect the
Figure 1. Intermediate severity LIS identical to the LIS1-associated phenotype in group 1. In this and other brain imaging figures, each row shows multiple
images from the same patient. The columns contain midline sagittal (far left column) and parasagittal (second column) images, axial images through the
deep nuclei (center column) and bodies of the lateral ventricles (fourth column) and coronal images through the hippocampus (far right column). When
coronal images were not available, low axial images through the cerebellum are shown instead. The horizontal white lines in the low posterior fossa in the
midline sagittal images (left column) indicate the expected level of the lower border of the vermis at the level of the obex. A set of normal control images
is shown in the top row of Supplementary Material, Figure S1. This figure shows three group 1 patients with the recurrent p.R402C TUBA1A mutation. All
three have frontal pachygyria and posterior agyria consistent with classic LIS grade 3 and a posterior more severe than anterior (p . a) gradient. Other features
include dysmorphic corpus callosum with small rostrum and genu plus a flattened anterior body, poorly myelinated and so unseen internal capsules and round
hippocampi with thick leaves [black arrowheads in (E), (J) and (O)]. One patient also has a mildly small cerebellar vermis [space between horizontal white line
and lower border of vermis in (A)]. This appearance is essentially identical to patients with heterozygous LIS1 null mutations and deletions. In addition, the
tectum appears mildly enlarged (arrowheads in (A), (F) and (K), which differs from other types of LIS. These images come from subjects LP95-073 (A–E),
LR07-008 (F–J) and LR08-035 (K–O).
2820Human Molecular Genetics, 2010, Vol. 19, No. 14
interaction between these helices. Because H11–H12 lie on the
interface between a-tubulin and various MAPs [e.g. kinesin
KIF1A (18), DCX (19), MAP2c (20), and Dynein (21)], the
mutations are also likely to affect the interaction between
tubulin and MAPs. The interaction network between H11 and
H12 is complex. First, the guanidinium ion of R402 is involved
in a cation–p interaction with the aromatic ring of Y399,
which in turn forms an H bond with S419-Og. Clearly, both
the R402H and the R402C mutations will abolish the cation–
p interaction, since neither histidine nor cysteine can be
involved in this type of interaction. Furthermore, because a
leucine side chain cannot form an H bond, the S419L mutation
cannot stabilize Y399 in the ideal position to form a cation–p
interaction with R402. Second, the guanidinium ion of R422
forms three H bonds with the carboxylate group of D396 and
a salt bridge with the carboxylate group of D392. Neither the
R422C nor the R422H mutations can form these interactions
(although histidine is able to form an H bond, it is too far
from D396). The M425K mutation might also interrupt this
network by competing with R422 for the interaction with
D392 (due to a change from a neutral residue to a positively
charged one). Finally, the L397P mutation is expected to intro-
duce a kink in H11 (since the cyclization of the proline side
chain prevents the regular backbone H bond formation),
which will affect the position of D396 and D392 relative to
R422 and possibly their interaction.
The tubulin gene family in humans is extensive, comprising at
least nine a, nine b, two g and single d and 1 subunits, all
expressed in different temporal, spatial and subcellular
locations. TUBA1A is one of three a-tubulin genes that
cluster on chromosome 12q13.12. The encoded protein is
formed by a core of two b-sheets surrounded by a-helices
and can be divided into three functional domains: (i) an
N-terminal domain (residues 1–205) that contains the
guanine nucleotide-binding region; (ii) an intermediate
domain (residues 206–381) containing the Taxol drug-binding
site and (iii) a C-terminal domain (resides 382–451) that
likely constitutes the binding surface for MAPs and molecular
motors such as kinesins and dyneins (17).
Mutation detection rates and testing recommendations
in LIS-SBH subtypes
We identified TUBA1A mutations in 17 patients, most (67%)
residing in the C-terminal domain. Five were detected in
patients with unexplained classic LIS (?7%), all at codon
R402. Another 10 mutations were found in patients with
LCH (?34%) scattered through most of the coding region
of the gene. In children with classic LIS resembling the
LIS1 (posterior predominate) pattern, the expected mutation
detection rate is ?85% (7). Our data predict that another
?1% of patients with classic LIS will have mutations of
TUBA1A (i.e. 7% of the remaining 15% of LIS yet
In children with LCH—a rare subtype of LIS—these results
increase the mutation detection rate from essentially nil up to
?30%, making TUBA1A the first gene to explain a significant
proportion of this rare phenotype. A few patients with another
LCH variant [LCH group b (5)] have mutations of RELN or
Although published reports (14,25) suggest that TUBA1A
mutations are found in some patients with SBH, we found
no mutations in a series of 22 patients with unexplained
Figure 2. Severe LIS resembling MDS in group 2. This figure shows two group 2 patients with the recurrent p.R402H TUBA1A mutation. Both have nearly
complete agyria consistent with classic LIS grade 1. Both also have dysmorphic corpus callosum with small rostrum–genu and flat anterior body and poorly
myelinated and so unseen internal capsules. The tectum again appears mildly enlarged [arrowheads in (A) and (F)], and both have a small cerebellar vermis
[note horizontal white line well below the lower border of the vermis in (A) and (F)]. The gyral malformation is essentially identical to MDS (due to deletion
17p13.3 that includes both LIS1 and YWHAE), while the cerebellar hypoplasia is more severe than most—but not all—children with MDS. These images come
from subjects LP97-039 (A–D) and LP97-041 (F–J).
Human Molecular Genetics, 2010, Vol. 19, No. 14 2821
SBH. We did see small areas partly resembling SBH in two
patients with the mild–moderate form of LCH (as shown in
Fig. 3N), but the brain imaging phenotype in these individuals
was clearly dominated by pachygyria, not SBH.
Importantly, we found one mutation in a girl with ACC and
CBLH but without LIS. This observation suggests that
mutations of TUBA1A may be responsible for a subset of chil-
dren with isolated ACC, isolated CBLH or—as in our
patient—combined ACC and CBLH without obvious LIS.
Further studies in these latter groups are clearly needed.
These results have significant implications for clinical mol-
ecular testing for LIS. We conclude that sequencing of
TUBA1A is so far indicated in two groups of patients. First
are those with classic LIS and negative deletion and sequen-
cing analysis for LIS1 and DCX. On brain imaging, we recog-
nize classic LIS based on the presence of extensive
agyria–pachgyria, 12–20 mm thick cortex and no more than
mild CBLH. Second are those with LIS and moderate-to-severe
small. Experts in brain imaging in this group of disorders could
exclude patients with a frontal gradient (LIS most severe in the
anterior frontal lobes) and patients with the rare pattern associ-
ated with RELN or VLDLR mutations (mild frontal pachgyria,
hippocampal hypoplasia and very small and near-afoliar cer-
ebellum), but we do not recommend excluding these patterns
generally. Although we did find one subject with combined
ACC and CBLH without LIS, we do not have sufficient data
to justify clinical testing of TUBA1A in children without LIS
at this time.
Our genotype–phenotype analysis revealed two major pheno-
type subsets, each with varying degrees of severity. The first
subset (groups 1 and 2) involves all seven patients with
mutations of codon R402, where the brain phenotype
matches the well-known phenotype associated with mutations
in LIS1 or combined LIS1 and YWHAE mutations. Specifi-
cally, the recurrent p.R402C phenotype resembles the LIS1
deletion or intragenic mutation phenotype, whereas the
p.R402H phenotype resembles MDS caused by the co-deletion
of LIS1 and YWHAE. The second subset (groups 3 and 4)
Figure 3. A novel and distinctive pattern of LCH in group 3. This figure shows three group 3 patients with TUBA1A missense mutations and a novel pattern of
malformations. All three have diffuse pachygyria or LIS grade 4 that appears mildly asymmetric and most severe over the central (mid- and posterior frontal,
perisylvian and anterior parietal) regions rather than over the posterior pole. One patient has a prominent layer of gray matter beneath the right perisylvian region
[arrow in (N)] that resembles SBH, but the overlying cortex appears thick so this is not true SBH. The hippocampi appear small and abnormally open and round
with thick leaves [arrowheads in (E) and (J)]. The basal ganglia are malformed, appearing as large round structures in which the caudate, putamen and globus
pallidus cannot be distinguished [asterisks in (C), (H) and (M)]. In the top row, the left basal ganglia appear to be located lateral rather than medial to the frontal
horn [asterisk in (C)]. Associated malformations include complete (A and K) or nearly complete (F) ACC, thin brainstem with flat pons [arrows in (A), (F) and
(K)], enlarged tectum [arrowheads in (A), (F) and (K)] and small cerebellar vermis [large space between the lower vermis and white lines in (A), (F) and (K)].
The cerebellar hemispheres are mildly small as well (O). These images come from subjects LR05-052 (A–E), LR08-340 (F–J) and LR07-244 (K–O).
2822 Human Molecular Genetics, 2010, Vol. 19, No. 14
involves patients with mutations of any other codon that differ
dramatically from the LIS1-associated pattern. Mutations of
other codons have much more variable phenotypes with
more variable severity and (anterior–posterior) gradient of
LIS and more consistent hypoplasia of the corpus callosum
The second subset includes children with the rare LCH phe-
notype. Ingroup 3, the gyral pattern often appeared most severe
in the middle (posterior frontal or perisylvian-parietal regions),
rather than at the frontal or occipital pole. Also, the callosal and
cerebellar defects varied substantially in severity. Finally, the
LCH seen in our group 4 is far more severe than ever seen in
patients with LIS1 or codon R402 mutations. Taken together,
our genotype–phenotype analysis suggests that mutations
involving R402 specifically disrupt the LIS1-associated neur-
onal migration pathway, whereas other mutations are likely to
disrupt multiple pathways and functions.
We reviewed the descriptions and available brain images for
other published patients with TUBA1A mutations and found
them generally compatible with our analysis of the phenotype
spectrum (11–14,25). This was particularly true for the
high-resolution images provided in Fig. 1 in the most recent
Mechanisms of action for TUBA1A mutations
The mechanism(s) leading to the LIS or LCH phenotypes with
heterozygous mutations is not clear, and we considered both
haploinsufficiency and a dominant-negative effect. Several
lines of evidence may support a dominant-negative mechan-
ism of action. First, our data and those of others (12,16)
show that mutant TUBA1A protein readily incorporates into
the cellular cytoskeletal network, rather than failing to incor-
porate as might be expected with loss of function. Second,
the observation of a nonsense mutation, frameshift mutation
or gene deletion in even a single patient would support hap-
loinsufficiency, but only missense mutations of TUBA1A
have been identified to date. To extend this observation, we
Figure 4. Group 4 consists of severe LCH. This figure shows three group 4 patients with heterogeneous TUBA1A missense mutations and the most severe form of
LCH. All three have a low forehead indicating microcephaly, marked thinning of the cortex and white matter with severe ventriculomegaly and nearly complete
agyria except for very limited pachygyria over the frontal poles. However, the appearance of the cerebral cortex differs between the three patients. In the first
patient, the ventriculomegaly is severe and the cerebral wall including the cortex is abnormally thin. In the first and third, the cortex is thick with a smooth lower
border [arrows in (C), (D), (N)]. In the second, the cortex is thick with an undulating almost sinusoidal lower border [arrows in (H) and (I)]. Compare this pattern
with subject LP99-059 shown in Fig. 6F in a prior publication (5). The hippocampi are very small and globular [arrowheads in (E), (J) and (O)]. The basal
ganglia are very small and dysplastic with no differentiation between caudate, putamen and globus pallidus [asterisks in (C), (H) and (M)]. Other changes
are also severe, including complete ACC, very thin brainstem with flat pons [arrows in (A), (F) and (K)], enlarged tectum [arrowheads in (A), (F) and (K)],
severe diffuse cerebellar hypoplasia [large space between lower border of the vermis and white line in (A), (F) and (K)] and relatively enlarged posterior
fossa. These overlapping patterns correspond to our prior LCH groups c and f (5). These images come from subjects LR05-388 (A–E), LR07-213 (F–J),
and LR08-388 (K–O).
Human Molecular Genetics, 2010, Vol. 19, No. 142823
searched several large cytogenetic databases including Deci-
bwhpathology.org/dgap/) and ECARUCA (http://agserver01.
azn.nl:8080/ecaruca/ecaruca.jsp), as well as PubMed, but
found no reports of individuals with TUBA1A deletions.
Further, no 12q13.12 deletions that contain TUBA1A were
orders screened by microarrays (Swaroop Aradhya, GeneDx;
Lisa Schaffer and Jill Rosenfeld, Signature Genomics, personal
communications, 2009). We cannot exclude the possibility that
think that the pathogenic mechanisms are more complex than
simple haploinsufficiency given that LIS-associated TUBA1A
mutations incorporate and maintain typical microtubule struc-
ture. These mutations could disrupt the binding sites for
MAPs, leading to altered function or possibly dominant-
Recurrent R402 mutations
Mutations involving the R402 residue have been reported in a
total of eleven patients to date [seven described here and four
elsewhere (13,14)], demonstrating that this codon is a muta-
tional hotspot in TUBA1A. Our modeling data show that the
R402 residue is located on a-helix H11, which lies at the
outer surface of microtubules at the interface between
a-tubulin and various MAPs and molecular motor proteins,
including kinesin KIF1A (18). Our cellular assays clearly
demonstrated that mutant R402 proteins are translated, loca-
lize to the cytoplasm and incorporate properly into the cyto-
skeletal network. Collectively,
mutations involving the R402 residue are more likely to
affect the interaction between tubulin and tubulin-binding
proteins than to affect overall tubulin architecture. However,
further biochemical experiments would be needed to substanti-
ate these predictions. Given that R402 mutations result in a
LIS1-like phenotype, we hypothesize that mutations involving
this residue specifically disrupt the LIS1 signaling pathway.
We propose that this may occur in a kinesin-dependent
manner that involves a LIS1-dynein-transportable microtubule
(tMT) complex, based on a recent observation that LIS1 and
cytoplasmic dynein co-migrate towards the plus end of micro-
tubules in a complex with tMT (26). In this model, LIS1 fixes
dynein on tMT (freighter tubulins), which are then transported
to the plus end of cytoskeletal MTs in a kinesin-dependent
manner. Thus, we hypothesize that the R402 mutation specifi-
cally interferes with kinesin-mediated anterograde transport of
LIS1-coupled dynein, without disrupting other functions of the
We further hypothesize that the LCH-associated mutations
of TUBA1A disrupt two or more distinct neuronal migration
pathways that converge on TUBA1A, such as LIS1-related
and DCX-related pathways, resulting in the chaotic LIS gradi-
ent and severe callosal and cerebellar phenotypes. It follows
that mutations at less critical residues might cause corpus cal-
losum and cerebellar malformations without obvious LIS, as
we now document in one patient.
In conclusion, we have identified numerous novel mutations
in TUBA1A, including several recurrent mutations at two
codons that result in homogenous phenotypes. We provide
mutationfrequency data thatwill contributetoclinical diagnos-
tics and demonstrate a wider spectrum of phenotypes than pre-
viously reported that now includes severe and moderate
classic LIS (groups 1 and 2), severe and moderate LCH
(groups 3 and 4) and ACC and CBLH not associated with LIS
(group 5). We also show that LIS-associated mutations are
likely to act in a dominant-negative manner by incorporating
Figure 5. Recombinant wild-type and mutant FLAG-tagged TUBA1A incorporate into the normal interphase microtubule network. After transfection into P19
cells, wild-type and mutant recombinant TUBA1A were visualized using methanol fixation and immunostaining using anti-FLAG antibodies.
2824Human Molecular Genetics, 2010, Vol. 19, No. 14
into microtubules and disrupting tubulin-MAP interactions.
Certainly, tubulin genes are emerging as strong candidates
for analysis in neuronal migration disorders, given recent
additional reports of mutations in TUBB2B in asymmetrical
polymicrogyria (27) and TUBA8 in polymicrogyria with optic
nerve hypoplasia (28).
MATERIALS AND METHODS
The Institutional Review Boards at the University of Chicago
and the University Hospital of Wales approved this study.
From our large cohort of subjects with unexplained LIS
documented by review of brain imaging (available for all
subjects and reviewed by W.B.D. or D.T.P.) and DNA
samples available (n ¼ 125), we selected 72 patients with
classic LIS (29 females and 43 males), 22 with SBH (13
females and 9 males), 29 with LCH (17 females and 12
males) most of whom also had partial or complete ACC
and two females with LIS and ACC but normal cerebellum
on imaging. We combined the latter two groups for further
analysis. All LIS subgroups were defined years before this
mutations, missense mutations, deletions or duplications in
LIS1 and DCX was negative in most patients (7). In the
remainder, the most appropriate gene was tested with nega-
tive results. Specifically, we tested only LIS1 in some
Figure 6. Mapping of TUBA1A mutations onto the 3D structure of kinesin KIF1A–microtubule complex. The KIF1A/microtubule complex: a-tubulin is shown
in blue ribbons, b-tubulin in dark and KIF1A in green [PDB ID: 2HXF (33)]. Residues mutated in TUBA1A are shown as light blue spheres; ADP and ATP
molecules in stick representation. Substitutions R402H/R402C, S419L, R422H/R422C, L397P and M425K on a-helices H11–H12 are likely to affect H11–H12
interactions, position, orientation and interactions with tubulin-binding proteins (e.g. KIF1A, DCX, MAP2c and Dynein). The guanidinium ion of R402 is
involved in a cation–p interaction with the aromatic ring of TUBA1A residue Y399, which in turn forms an H bond with S419-Og. Both the R402H and
R402C mutations will abolish the cation–p interaction, since neither histidine nor cysteine can be involved in this type of interaction. Because a leucine
side chain cannot form an H bond, the S419L mutation cannot stabilize Y399 in the ideal position to form the cation–p interaction with R402. The guanidinium
ion of R422 forms multiple H bonds with the carboxylate group of D396 and a salt bridge with the carboxylate group of D392. Neither the R422C nor R422H
mutations can participate in these interactions (although histidine is able to form an H bond, it is too far from D396). The M425K mutation might also interrupt
this network by competing with R422 for the interaction with D392 (due to a change from a neutral residue to a positively charged one). The L397P mutation is
expected to introduce a kink in H11, which will affect the position of D396 and D392 relative to R422. C: The N329S mutation is located on a-helix H10 at the
interface between a - and b-tubulin subunits, close to the GTP binding site. The interaction of H10 with a-tubulin is stabilized by the side chains of residues
N329 and K326. The N329 forms two H bonds, one with D179 and the other with V177. The N329S mutation will not allow the forming of the H bonds with
V177 and therefore is likely to destabilize interactions at the a–b interface. H bonds are marked in black and salt bridges in gray.
Human Molecular Genetics, 2010, Vol. 19, No. 14 2825
patients with posterior-predominant LIS and tested DCX only
in some with anterior-predominant LIS. We subsequently
added two patients in whom TUBA1A mutations were
found in clinical laboratories (one unexpectedly given the
lack of LIS) and referred to us.
DNA isolation, amplification and sequencing
DNA was extracted from leukocytes from EDTA-treated
blood using either the Puregene kit (Gentra Systems, Inc.,
Minneapolis, MN, USA) or the MagNAPure Total Nucleic
Acid Extraction system (Roche Diagnostics, Indianapolis,
IN, USA) following the manufacturer’s protocols. PCR-
amplification primers were designed using Primer3 (http
://frodo.wi.mit.edu/) with M13 forward and reverse tails
added to each primer to facilitate high-throughput DNA
sequencing (Table 1). DNA was amplified in a reaction com-
prising: 20 ng genomic DNA, 1× buffer I (1.5 mM MgCl2,
Applied Biosystems, Foster City, CA, USA), 1 mM dNTPs
(Applied Biosystems), 0.4 mM primer (each of forward and
reverse; IDT, Coralville, IA, USA) and 0.25 U AmpliTaq
Gold (Applied Biosystems) in a total volume of 10 ml. Ther-
mocycling conditions were as follows: 948C for 10 min; 35
cycles of 948C for 30 s, annealing temperature (53–608C)
for 30 s and 728C for 30 s and final extension of 728C for
10 min. Variations in reaction composition and cycling con-
ditions were required for a small number of amplicons. PCR
products were purified in a 10 ml reaction comprising 6.6 U
Exonuclease I and 0.66 U shrimp alkaline phosphatase that
were incubated at 378C for 30 min followed by 808C for
15 min. Sequencingreactions
BigDye terminators on an ABI 3730XL 96-capillary auto-
mated DNA sequencer (Applied Biosystems) at The Univer-
sity of Chicago DNA Sequencing and Genotyping Core
Facility. Sequence data were imported as ABI files into
Mutation Surveyor v3.10 (SoftGenetics, State College, PA,
USA). Sequence contigs were assembled by aligning ABI
files against GenBank reference sequence files obtained
from the National Center for Biotechnology Information
putative mutations, the entire length of the sample trace
was manually inspected for quality and variation from the
reference trace. All detected variants were visually reviewed
by two trained individuals and were confirmed using
Mutations were introduced into the pRK5TUBA1A-CFLAG
expression construct using the Quickchange site-directed
C-terminally FLAG-tagged TUBA1A under the control of
the CMV promoter. Wild-type and mutant constructs were
transfected into P19 cells (ECACC, 95102107) using Lipofec-
tamine LTX reagent (Invitrogen). After 24 h, cells were fixed
in ice-cold methanol, permeabilized with 0.1% (v/v) Triton
X-100 and stained with antibodies recognizing FLAG
(Sigma) and secondary anti-mouse antibodies conjugated to
Alexa 488 (Molecular Probes).
Structural modeling of TUBA1A mutations
Two atomic models of tubulin complexes were used for the
analysis of the mutations: (a) protofilaments of a- and
microtubule complex. The former model was based on
docking of the a- and b-tubulin heterodimer crystal structure
(PDB id: 1JFF) (31) into the cryo electron microscopy
(cryoEM) map of the intact microtubules at 8 A˚resolution
(32) (coordinates kindly provided by Ken Downing, Life
Sciences Division, Berkeley Lab), while the latter was based
on docking of the same structure (PDB id: 1JFF) in addition
to the kinesin KIF1A motor domain crystal structure (PDB
id: 1VFV) into the cryoEM map of the complex in an
AMPPNP (5′-adenylyl-b,g-imidodiphosphate) form at 10 A˚
resolution (PDB id: 2HXF) (33). In the original models, the
interfaces between the domains suffer from minor clashes,
most likely due to conformational differences between the
structures of the domains in their isolated form and in the
context of their assemblies. To remove these clashes, we
first added hydrogens using VMD (34) and then applied con-
jugate gradient energy minimization to the interface between
two heterodimers of a- and b-tubulin in the first model and
to the interface between KIF1A and a-tubulin in the second
model. The minimization was performed using NAMD (35)
with the CHARMM27 forcefield (36). The final models were
visualized and analysed with chimera (37); mutations were
introduced based on the Dunbrack backbone-dependent
rotamer library (38) and H bonds were identified with the
FindHBond method (39). Cation–p interactions were ident-
ified using the program CaPTURE (40).
(b) the kinesin KIF1A-
Supplementary Material is available at HMG online.
We thank the parents of the many children with LIS who have
allowed us to include them in our studies over the past 25
Conflict of Interest statement. None declared.
This study was supported in part by a grant from the NIH to
W.B.D. (1P01-NS039404 and 1R01-NS050375), the Wales
Office of Research and Development (M.I.R. and T.D.C.)
and the Medical Research Council (K.H., R.J.H. and M.T.).
We thank Autism Speaks for granting a Postdoctoral Fellow-
ship Award to R.A.K. Funding to pay the Open Access
Charge was provided by the National Institutes for Health.
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