Lissencephaly and the molecular basis of neural migration

Abstract

Migration of post-mitotic neurons from the ventricular zone to the cortical plate during embryogenesis comprises one of the most critical stages in brain development. Deficiency of this process often results in major brain malformations, including human lissencephaly (smooth brain). Since discovery of the first genetic cause of lissencephaly, deletions of chromosome 17p13.3 in Miller-Dieker syndrome, rapid progress in our understanding of neuronal migration has been made based on advances in both brain imaging technology and molecular genetics. This progress has resulted in a new system of classification that began with pathological descriptions and has evolved to include patterns on brain imaging, causative genes and most recently the molecular pathways and proposed modes of migration involved. In this review, we summarize current knowledge regarding five genes that cause or contribute to human lissencephaly, including LIS1, 14-3-3 epsilon, DCX, RELN and ARX. Each of these is associated with a characteristic pattern of malformation that involves the cerebral cortex and sometimes other brain structures. Based on detailed genotype-phenotype analysis, we can now infer the most likely causative gene based on brain imaging and other clinical findings, and inversely are becoming able to predict clinical severity based on the specific mutations detected. We also hypothesize, for the first time, a relationship between the specific type of lissencephaly observed and deficiency of specific modes of neuronal migration.

Full text

Lissencephaly and the molecular basis of
neuronal migration
Mitsuhiro Kato
1,4
and William B. Dobyns
1– 3,
*
1
Department of Human Genetics,
2
Department of Neurology and
3
Department of Pediatrics,
The University of Chicago, Chicago, IL 60637, USA and
4
Department of Pediatrics,
Yamagata University School of Medicine, Yamagata, Japan
Received January 17, 2003; Revised and Accepted February 5, 2003
Migration of post-mitotic neurons from the ventricular zone to the cortical plate during embryogenesis
comprises one of the most critical stages in brain development. Deficiency of this process often results in
major brain malformations, including human lissencephaly (smooth brain). Since discovery of the first
genetic cause of lissencephaly, deletions of chromosome 17p13.3 in Miller–Dieker syndrome, rapid progress
in our understanding of neuronal migration has been made based on advances in both brain imaging
technology and molecular genetics. This progress has resulted in a new system of classification that began
with pathological descriptions and has evolved to include patterns on brain imaging, causative genes and
most recently the molecular pathways and proposed modes of migration involved. In this review, we
summarize current knowledge regarding five genes that cause or contribute to human lissencephaly,
including LIS1, 14-3-3e, DCX, RELN and ARX. Each of these is associated with a characteristic pattern of
malformation that involves the cerebral cortex and sometimes other brain structures. Based on detailed
genotype–phenotype analysis, we can now infer the most likely causative gene based on brain imaging and
other clinical findings, and inversely are becoming able to predict clinical severity based on the specific
mutations detected. We also hypothesize, for the first time, a relationship between the specific type of
lissencephaly observed and deficiency of specific modes of neuronal migration.
INTRODUCTION
Migration of post-mitotic neurons from the ventricular zone to
form the cortical plate comprises one of the most critical stages
in brain development. Our understanding of this complex
process has progressed based on studies of human malformations
and mouse mutants with deficient neuronal migration, particu-
larly the malformation known as lissencephaly (LIS) or ‘smooth-
brain’. LIS is characterized by a smooth or nearly smooth
cerebral surface. It encompasses a spectrum of gyral malforma-
tions from complete agyria (absent gyri) to regional pachygyria
(broad gyri), and merges with subcortical band heterotopia
(SBH). LIS is always associated with an abnormally thick cortex,
reduced or abnormal lamination and diffuse neuronal heterotopia
(1,2). SBH or ‘double cortex’ consists of circumferential bands
of heterotopic neurons located just beneath the cortex and
separated from it by a thin band of white matter (3,4).
Several different types of LIS have been recognized. The
most common type, known as classical LIS (previously type I),
has a very thick 10–20 mm cortex and no other major brain
malformations. This is the only type associated with SBH. Less
common types are associated with agenesis of the corpus
callosum (ACC) or severe cerebellar hypoplasia (5,6).
In this paper, we review recent discoveries regarding the
molecular mechanisms that regulate or effect neuronal
migration to the cerebral cortex, emphasizing those derived
from studies in humans with LIS. Other genes and proteins
identified in humans with periventricular nodular heterotopia
or cobblestone complex malformations (previously type
II LIS), or in mouse mutants have been reviewed elsewhere
(7–11).
HUMAN LISSENCEPHALY GENES
To date, five genes have been identified that cause or contribute
to LIS in humans: LIS1, 14-3-3e, DCX, RELN and ARX. Their
respective subtypes and syndromes are shown in Figures 1
and 2, and can usually be distinguished based on detailed
analysis of the phenotype as shown in Figure 3.
*To whom correspondence should be addressed at: The University of Chicago, Department of Human Genetics, Room 319 CLSC, 920 E. 58th Street,
Chicago, IL 60637, USA. Tel: þ 1 7738343597; Fax: þ1 7738348470; Email: wbd@genetics.bsd.uchicago.edu
Human Molecular Genetics, 2003, Vol. 12, Review Issue 1 R89–R96
DOI: 10.1093/hmg/ddg086
Human Molecular Genetics, Vol. 12, Review Issue 1 # Oxford University Press 2003; all rights reserved

LIS1
LIS1 or PAFAH1B1 was the first human neuronal migration
gene to be cloned, and encodes the non-catalytic alpha subunit
of the intracellular Ib isoform of platelet-activating factor
acetylhydrolase (12,13). The gene is located in human
chromosome 17p13.3 and consists of 11 exons with a coding
region of 1233 bp. LIS1 protein is expressed predominantly in
fetal and adult brain (14), and interacts with tubulin to suppress
microtubule dynamics (15). It is a highly conserved protein
with near-identity between mouse and human, and 42%
homology to NudF, an ortholog found in Aspergillus
nidulans (16). Studies of NudF and related genes such as
NudE have shown that LIS1 participates in cytoplasmic
dynein-mediated nucleokinesis, somal translocation and cell
motility (17) as well as mitosis (neurogenesis) and chromo-
some segregation (18). In animal experiments including human
cells, Lis1 acts via a signaling pathway that includes NudE,
NudeL, cytoplasmic dynein, dynactin, and CLIP-170 (19–26).
14-3-3e
This gene belongs to the 14-3-3 family of proteins that bind
to phosphoserine and phosphothreonine motifs in a wide
variety of proteins (27–31). It is also located in chromosome
Figure 1. Axial T
1
-(B, C, E, H) and T
2
-(A, D, F, G) weighted magnetic resonance images at the level of basal ganglia in five types of lissencephaly. In contrast to
a normal control (H), all types of LIS have broad or absent gyri and abnormally thick cortex, except for LIS grade 6 or SBH, in which the sulci separating gyri are
very shallow. The anterior to posterior or rostro-caudal gradient of LIS is strictly correlated with the causative gene. Specifically, mutations of DCX or RELN result
in an anterior more severe than posterior (a > p) gradient (A–D), while mutations of LIS1 with or without 14-3-3e or ARX lead to a posterior more severe than
anterior (p > a) gradient (E–G). The absolute thickness of the cortex and presence of a cell sparse zone also differ based on the causative gene. In patients with
mutations of DCX (A, B) or LIS1 (E, F), the cortex is very thick, typically 10–20 mm, and prominent cell sparse zones are seen in areas of agyria (arrowheads in A,
E, F). In patients with LIS with cerebellar hypoplasia group b [C, which resembles patients with known RELN mutations (67,94), although a mutation has not been
demonstrated in this patient] or ARX (G) mutations, the cortex is only moderately thick, typically 5–10 mm, and cell sparse zones are never seen, even in areas of
agyria (G). In LIS with cerebellar hypoplasia group b with or without proven RELN mutations, other images show an abnormal hippocampus and severe cerebellar
hypoplasia (not shown). In males with X-linked lissencephaly with abnormal genitalia due to ARX mutations, other images demonstrate poorly demarcated basal
ganglia often with small cysts, immature white matter, and agenesis of the corpus callosum (not shown). In heterozygous females, mutations of DCX result in SBH
(D), while mutations of ARX often result in agenesis of the corpus callosum (not shown). ILS, isolated lissencephaly; LCHb, lissencephaly with cerebellar hypo-
plasia group b; MDS, Miller–Dieker syndrome; XLAG, X-linked lissencephaly with abnormal genitalia.
R90 Human Molecular Genetics, 2003, Vol. 12, Review Issue 1

17p13.3, about 40 kb telomeric to LIS1, and has six exons
that encode 255 amino acids. The gene codes for a highly
conserved protein that binds to and protects phosphorylated
NUDEL from dephosphorylation by protein phosphatase
2A (PP2A). It is required for NUDEL localization and
cytoplasmic dynein function, and appears to be important
for neuronal migration based on mouse studies (Tokyo-oka
et al., submitted).
DCX
DCX or XLIS was first identified as the gene causing X-linked
LIS and SBH (32,33). It is located in chromosome Xq22.3–q23
and has nine exons (six coding exons) that code for a 360
amino acid protein. The DCX protein contains two tandem
evolutionarily conserved repeats (doublecortin domain) that
form a b-grasp superfold. Each of the repeat binds to tubulin
but not to microtubules, so that both repeats are necessary for
microtubule polymerization and stabilization (34–37), and pos-
sibly for direct interactions with LIS1 (38). DCX is expressed
exclusively in fetal brain, including forebrain and cerebellum.
RELN
Reln was cloned as the causative gene for the reeler mutant
mouse, which has abnormal lamination of the cerebral and
cerebellar cortices including inversion of the normal ‘inside-
out’ pattern found in mammals (39,40). The human gene is
located in chromosome 7q22 and consists of 65 exons covering
more than 400 kb of genomic sequence. It encodes a large
extracellular matrix protein with 3460 amino acids that is
secreted by Cajal–Retzius cells in the preplate (41) and has
94.2% homology with the mouse ortholog (42). Reln functions
in a signal transduction pathway via the apolipoprotein E2
Figure 2. Correlation of LIS grade and gradient and associated brain malformations with causative genes. LIS grades 1 and 2 consist of diffuse agyria, although
patients with LIS grade 2 have a few shallow sulci over the frontal or occipital pole. LIS grade 3 comprises mixed agyria and pachygyria, grade 4 pachygyria only,
grade 5 mixed pachygyria and SBH, and grade 6 SBH only. The most severe form of LIS (grade 1) is typically caused by either combined LIS1 and 14-3-3e
deletion or a severe mutation of DCX. Mutations of LIS1 alone typically cause LIS grades 2–4, most often grade 3, with very rare patients having posterior
SBH. Mutations of DCX cause a wide range of LIS, although grade 3 has proven to be rare. Mutations of RELN cause both cerebral (LIS) and cerebellar mal-
formations, which resembles the defects found in the reeler mouse. Mutations of ARX most often result in LIS grade 3, in addition to the basal ganglia, white
matter and callosal abnormalities. At least half of all female carriers of ARX mutations that cause XLAG in males have ACC. Black box, most frequently observed;
dark grey box, sometimes observed; light grey box, rarely observed. CBLH, cerebellar hypoplasia. The asterisk indicates it may result from somatic mosaicism.
Human Molecular Genetics, 2003, Vol. 12, Review Issue 1 R91

(ApoER2) and very low-density lipoprotein receptors (Vldlr)
(43,44), which activate the downstream cytoplasmic protein
Dab1 (45,46). The brains of mutant mice strains with
disruptions of mDab1 or of both ApoER1 and Vldlr closely
resemble the brain of the reeler mouse (44,47).
ARX
ARX is a paired-class homeobox gene that shows significant
homology with the Drosophila al (aristaless) gene in the
homeodomain and C-peptide or aristaless domain (48). The
gene is located in human chromosome Xp22.13 and consists of
five exons that encode a protein of 562 amino acids (49,50).
ARX is specifically expressed in interneurons of the forebrain
and in the interstitium of the male gonad (48,50). It is involved
in differentiation of the testes and the embryonic forebrain,
especially in proliferation of neural precursors and differentia-
tion and tangential migration of interneurons (50).
GENOTYPE–PHENOTYPE CORRELATION
WITH MUTATIONS OF KNOWN
LISSENCEPHALY GENES
LIS1
The most important characteristics of LIS in patients with LIS1
mutations are the very thick 10–20 mm cortex, gyral mal-
formations that are more severe in posterior than anterior (p > a
gradient) brain regions (51,52) and prominent cell sparse zone
in the cortex. The corpus callosum and cerebellum appear
normal or mildly hypoplastic on brain MRI (6,52).
Mutations of LIS1 cause isolated lissencephaly sequence
(ILS) or rarely isolated SBH, both with a clear p > a gradient.
We found a significant correlation between LIS–SBH severity
and mutation type in these disorders. Patients with submicro-
scopic deletions of 17p13.3 that include LIS1 and those with
null mutations in the coiled coil (MAP1B homology) domain
in exons 2–5 usually had LIS grade 2–3, with a mean grade of
2.43 for the latter group (52,53). Patients with null mutations
distal to the coiled coil domain usually had LIS grades 3–4,
with a mean grade of 3.18. Patients with missense mutations of
LIS1 had even less severe LIS unless a critical amino acid
residue was involved, with a mean grade of 4.20 (53–55).
All but one of the missense mutations are associated with
milder phenotypes than patients with null mutations, which
suggests some residual LIS1 protein function. However, in vitro
assays show that all mutant proteins completely lose the
capacity to interact with NUDE as well as the 29 and 30 kDa
subunits of platelet activating factor acetylhydrolase,
PAFAH1B2 and PAFAH1B3 (22,56). Thus, the basis for
differing severity among patients with missense mutations is
unclear. The mildest LIS1-associated phenotype known con-
sists of infrequent seizures, mild clumsiness and normal
intelligence with pachygyria limited to the posterior parietal
and occipital lobes. The affected boy had a missense mutation
in the second WD repeat resulting in substitution of serine for
glycine (G162S). This is predicted to be a mild mutation, as
serine is found at the same position in other WD family
proteins (55,57).
Figure 3. Flow chart to predict the most likely causative gene in patients with LIS by analysis of brain MRI.
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LIS1 and 14-3-3e
The association between LIS and deletions of 17p13.3 was first
recognized in patients with Miller–Dieker syndrome (MDS),
which consists of LIS and facial abnormalities including
prominent forehead, bitemporal hollowing, short nose with
upturned nares, prominent upper lip with downturned vermi-
lion border and small jaw, and sometimes other congenital
anomalies (58,59). Our previous studies suggested that patients
with MDS have more severe LIS than patients with ILS, and
that deletions in MDS extend further toward the 17p telomere
than in ILS, suggesting that another gene involved in brain
development is located distal to LIS1 (52,60).
We recently completed a contig across most of this 400 kb
region, and studied 30 patients with MDS or ILS and deletions of
the region with a set of FISH probes and somatic cell hybrids
(61). MDS was always associated with LIS grade 1 (essentially
complete agyria), and the deletion in MDS extends from LIS1 to
include all or part of BAC RPCI11-818O24, which contains the
CRK and 14-3-3e genes. The 14-3-3e gene is the best candidate
to account for the more severe LIS phenotype in MDS, as the
mouse knockout has mild defects of neuronal migration, although
a Crk knockout has not yet been reported. As would be predicted
from our results in humans, mice compound heterozygous for
14-3-3e and Lis1 display more severe brain defects than either
heterozygous mutant mouse alone (Toyo-oka et al.,submitted).
DCX
The most important characteristics of LIS in patients with
DCX mutations are the very thick 10–20 mm cortex, gyral
malformations that are more severe in anterior than posterior
(a > p gradient) brain regions, prominent cell sparse zone and
the occurrence of SBH rather than LIS in heterozygous females
(3,51,52). Hypoplasia of the cerebellar vermis is sometimes
associated with LIS caused by either LIS1 or DCX mutation,
classified as LIS with cerebellar hypoplasia group a (6,52).
In contrast to LIS1, missense mutations of DCX are more
common than truncations. However, genotype–phenotype
correlation is complex. Our experience has shown that the
same (presumably severe) mutations that cause LIS grade 1 or
agyria in males cause diffuse thick SBH in females. Similarly,
the same (presumably mild) mutations that cause LIS grade 4
or frontal pachygyria in males cause diffuse thin or partial
frontal SBH in females. A few missense mutations have led to
partial frontal SBH in males and either very mild or normal
phenotypes in their carrier mothers, who had germline
mutations and random X inactivation (54,62).
Despite substantial experience, predicting the phenotype
based on the genotype remains difficult due to incomplete
knowledge regarding function of specific amino acids within
the two microtubule-binding or doublecortin domains in exons
4–6, and relatively frequent post-zygotic mosaicism. Our recent
data have shown that truncation mutations in all but the last two
exons (exons 8 and 9) cause a severe phenotype, while
truncation mutations in exon 9 cause a variable phenotype
suggesting nonsense-mediated mRNA decay in DCX tran-
scripts. No truncations in exon 8 have been reported. Missense
mutations of DCX cluster in the doublecortin domains in exons
4–6, where they cause a variable phenotype (36,37,51,63).
Missense mutations in exons 7–9 have never been observed,
and so presumably cause a very mild or no phenotype (63).
Finally, post-zygotic mosaicism appears to be an important
mechanism in both sexes. In males, post-zygotic mosaicism
ameliorates the phenotype, resulting in SBH or mild LIS
(grades 5 and 6) rather than severe LIS (grade 1 or 2) (64–66).
In females, post-zygotic mosaicism has been found in mothers
of several affected patients (64). Owing to the difficulty in
detecting mosaicism in heterozygous females, a high index of
suspicion is required, and must be taken into account when
providing genetic counseling.
RELN
Mutations of RELN have been reported in six children with a
LIS variant from two unrelated families (67). Both families
showed exon skipping resulting in undetectable or reduced
levels of RELN protein. The same pattern of LIS was described
in four patients from two unrelated Japanese families, one of
whom had reduced levels of RELN protein in serum (6,68).
These patients have been classified as LIS with cerebellar
hypoplasia group b (6). In these children, the malformation is
characterized by a moderately thick 5–10 mm cortex, LIS that
appears more severe in anterior than posterior (a > p gradient)
brain regions, malformed hippocampus and very small
cerebellum virtually lacking folia.
ARX
Mutations of ARX cause a wide range of phenotypes that
correlate closely with the type of mutation. Hemizygous males
with null and non-conservative missense mutations have
a well-delineated syndrome known as X-linked lissencephaly
with abnormal genitalia (XLAG) (5). The most important
characteristics of LIS in patients with XLAG are a moderately
thick 5–10 mm cortex, gyral malformations that are more
severe in posterior than anterior (p > a gradient) brain regions,
deficiency of small granular neurons throughout the cerebral
cortex, abnormal signal of white matter, ACC, and cystic or
fragmented basal ganglia (50,69) (Kato et al., submitted). Rare
patients with null mutations or missense mutation in the
homeobox have a variant with hydranencephaly or isolated
ACC, both with abnormal genitalia (Kato et al., submitted).
Missense or in frame expansion mutations of ARX cause
familial X-linked infantile spasms or West syndrome, sporadic
cryptogenic or non-symptomatic West syndrome, X-linked
myoclonic epilepsy with spasticity and mental retardation,
Partington syndrome (mental retardation and dystonia), non-
syndromic X-linked mental retardation, and possibly autism
(49,70–74) (Kato et al., submitted). We also found several
females with symptomatic or asymptomatic ACC, who were
carriers of the same ARX mutations that caused XLAG in
males. Thus, mutations of ARX are clearly associated with
remarkable pleiotropy that includes seemingly disparate
phenotypes both with and without malformations.
LIS AND MECHANISMS OF MIGRATION
For many years, neuronal migration along radial glial fibers has
been the most widely accepted mechanism of cortical
Human Molecular Genetics, 2003, Vol. 12, Review Issue 1 R93

formation, and disturbance of neuronal migration the cause of
LIS (2,75,76). However, recent studies have demonstrated three
different modes of migration, including two forms of radial
migration (somal translocation and glia-guided locomotion), as
well as tangential or non-radial migration (77–79).
Somal translocation consists of movement of the soma and
nucleus toward the cortical plate in a long, radially oriented
basal process of the cell that terminates at the pial surface
(79,80). Glia-guided migration consists of slower movement
along the scaffold of radial glial fibers. In general, early-
generated cells such as preplate neurons use somal trans-
location only. Later-migrating pyramidal neurons first use
glia-guided locomotion and subsequently somal translocation
as they move past earlier-generated neurons to form the ‘inside-
out’ pattern of the mammalian neocortex (80,81). Tangential
migration is used by GABAergic neurons to migrate from the
ventral to the dorsal telencephalon along corticofugal fibers. On
reaching the dorsal telencephalon, the cells change direction to
migrate into the cortex along either corticofugal projection
fibers or radial glial fibers (50,82–84). Some of these cells
briefly migrate toward the ventricular zone, before reversing
direction to migrate into the neocortex (85).
From the limited data available so far, it appears likely that
the three known modes of migration are affected in different
ways in the various LIS subtypes. In the cerebral cortex of
reeler mice (Reln deficiency), the preplate appears to form
normally, but later-migrating neurons fail to split the preplate
into marginal zone and subplate. Instead, they form layers
below the preplate with a reverse ‘outside-in’ pattern compared
with normal cortex (39,86,87). Reln is also required for the
formation of the radial glial scaffold in the hippocampus (88).
These observations suggest a role for Reln, and presumably
human RELN, in glia-guided locomotion.
As mentioned above, LIS1 interacts with tubulin and
participates in nucleokinesis and extension of the leading
process as well as mitosis (10). In compound heterozygous Lis1
mutant mice, splitting of the preplate is defective, leaving a
broad and poorly defined subplate, and cortical lamination is
completely disrupted as in humans with LIS (89). In vitro
studies of Lis1-deficient cerebellar granule cells demonstrate
deficient migration along neurites of other cells in culture (90).
While available data is less clear than for Reln, the abnormality
of the preplate and the severe, and thus most likely early-onset,
cortical disruption seem to implicate a defect of somal
translocation. The defective migration along neurites suggests
that migration along radial glia may also be deficient. Based on
these results, we hypothesize that both somal translocation and
glia-guided migration are disrupted by mutations of LIS1.
Studies of tangential migration have not yet been reported. The
remarkably thick cortex also supports a more severe defect of
migration with LIS1 compared to RELN mutations.
DCX is a microtubule-associated protein that may interact
with LIS1, and mutations cause severe LIS, similar to that
observed with LIS1 mutations (34,35,91). However, its function
remains poorly understood. Expression of DCX in both radial
columns and tangentially directed neurons of human fetal
cortex suggests that it may be involved in both radial and
tangential migration (92).
Arx is expressed in the ganglionic eminences and the
neocortical ventricular zone, so both radial and tangential
migration could be affected. However, the data available to date
implicate primarily tangential migration. In Arx mutant mice,
mutant GABAergic interneurons originating from the gang-
lionic eminence remain near the subplate for at least 3 days,
and then migrate to the cortex where they are aberrantly
scattered throughout the cortical plate (50). In humans with
ARX mutations, the cortex contains almost exclusively
pyramidal neurons (69), which also implicates tangential
migration. The different pathological findings between humans
and mice may reflect differences in the origin of GABAergic
interneurons (93). We therefore hypothesize that ARX
deficiency results in defects primarily of tangential migration.
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