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Nonsyndromic mental retardation and cryptogenic epilepsy in women with Doublecortin gene mutations.

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Nonsyndromic Mental Retardation and
Cryptogenic Epilepsy in Women
with Doublecortin Gene Mutations
Renzo Guerrini, MD,1,2 Francesca Moro, PhD,2 Eva Andermann, MD,3 Elaine Hughes, MD,4
Daniela D’Agostino, MD,3 Romeo Carrozzo, MD,2 Andrea Bernasconi, MD,3 Frances Flinter, MD,5
Lucio Parmeggiani, MD,2 Anna Volzone, MD,2 Elena Parrini, BS,2 Davide Mei, MT,2 Jozef M. Jarosz, MD,6
Robin G. Morris, MD,7 Polly Pratt, PhD,7 Gaetano Tortorella, MD,8 François Dubeau, MD,3
Frederick Andermann, MD,3 William B. Dobyns, MD,9 and Soma Das, PhD9
DCX mutations cause mental retardation in male subjects with lissencephalypachygyria and in female subjects with
subcortical band heterotopia (SBH). We observed four families in which carrier women had normal brain magnetic
resonance imaging (MRI) and mild mental retardation, with or without epilepsy. Affected male subjects had SBH or
pachygyria-SBH. In two families, the phenotype was mild in both genders. In the first family, we found a tyr138his
mutation that is predicted to result in abnormal folding in the small hinge region. In the second family, we found an
arg178cys mutation at the initial portion of R2, in the putative ␤-sheet structure. Carrier female subjects with normal
MRI showed no somatic mosaicism or altered X-inactivation in lymphocytes, suggesting a correlation between mild
mutations and phenotypes. In the two other families, with severely affected boys, we found arg76ser and arg56gly
mutations within the R1 region that are predicted to affect DCX folding, severely modifying its activity. Both carrier
mothers showed skewed X-inactivation, possibly explaining their mild phenotypes. Missense DCX mutations may manifest as non-syndromic mental retardation with cryptogenic epilepsy in female subjects and SBH in boys. Mutation
analysis in mothers of affected children is mandatory, even when brain MRI is normal.
Ann Neurol 2003;54:30 –37
Lissencephaly and subcortical band heterotopia (SBH)
are brain malformations caused by deficient neuronal
migration. Affected individuals present with epilepsy
and mental retardation of variable severity. Both lissencephaly and SBH have been associated with mutations
of the LIS1 and DCX (doublecortin) genes.1–5
LIS1 mutations cause more severe malformations
posteriorly, whereas DCX mutations result in more severe malformations anteriorly.6,7 Mutations of DCX
have been described in sporadic male subjects with lissencephaly and female subjects with SBH, and in families with multiple affected subjects.2,8 –12 Heterozygous
female subjects from these families have thin frontal
SBH, although a few have had diffuse bands.12 Differ-
ences in phenotype have been attributed to both the
type of mutation and somatic mosaicism.9,11,12
We describe four families segregating missense mutations of DCX. In each family, one or more carrier
women had normal brain magnetic resonance imaging
(MRI), mild mental retardation, or borderline IQ, with
or without epilepsy. They were ascertained through
their affected sons or brothers who had pachygyriaSBH. Molecular studies indicated that the mechanisms
leading to seemingly normal neuronal migration in carrier women may result from either only mild loss of
function or skewed X-inactivation.
The possibility of carrier female subjects with mild
nonsyndromic mental retardation or epilepsy, means
From the 1Division of Child Neurology and Psychiatry, University of
Pisa; 2Research Institute, IRCCS Fondazione Stella Maris, Pisa, Italy;
3
Department of Neurology and Human Genetics, Montreal Neurological Hospital and Institute, Montreal, Canada; 4Department of
Paediatric Neurology, King’s College Hospital; 5Department of Clinical Genetics, Guy’s Hospital; 6Department of Neuroradiology, King’s
College Hospital; 7Department of Neuropsychology, Institute of Psychiatry, RG Mand King’s College Hospital, London, United Kingdom; 8Department of Medical and Surgical Paediatric Sciences, University of Messina, Messina, Italy; and 9Department of Human
Genetics, University of Chicago, Chicago, IL.
Received Sep 24, 2002, and in revised form Feb 10, 2003. Accepted
for publication Feb 11, 2003.
30
Address correspondence to Dr Guerrini, Division of Child Neurology and Psychiatry, IRCCS Fondazione Stella Maris University of
Pisa, Via dei Giacinti 2 56018, Calambrone, Pisa, Italy.
E-mail: renzo.guerrini@inpe.unipi.it
© 2003 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
that mutation analysis in mothers of affected children
is important, even when brain MRI is normal.
Subjects and Methods
Subjects
Clinical data were collected from patients, their parents, and
other relatives. Pedigrees were constructed extending back as
many generations as possible (Fig 1).
Family 1, with 23 living members, originated in Kent,
Southern England. Family 2, with 6 living members, originated from Sicily, Italy, and Family 3, with 5 living members, originated from London, UK. In these families, male
probands were referred because of developmental delay and
seizures. Family 4, with 24 living members, was of French
Canadian origin. The proband was referred because of seizures.
After informed consent, blood samples for mutation analysis were obtained from all personally examined subjects (see
Fig 1).
Magnetic Resonance Imaging
FAMILY 1. MRI was performed in six subjects at 1.5T
(IV:2, IV:3, IV:4, IV:5, IV:7, and IV:8) and in one at 1.0 T
(III:4). All subjects had axial fast spin-echo (SE) T2-weighted
images with 5mm thickness. Four (IV:2, IV:3, IV:5, IV:7)
had coronal fast SE T2-weighted scans with 3.0 to 3.5mm
thickness and volumetric T1-weighted gradient-echo (GE)
scans with coronal 1.5mm partitions and fluid-attenuated inversion recovery (FLAIR) images. Two had coronal 6mm SE
T1-weighted scans (IV:7 and IV:8). One (III:4) had additional coronal SE T1-weighted and 3mm coronal STIR images.
The proband and his mother were studied with
1.5T fast spoiled gradient echo (FSPGR), FLAIR, and
gradient-echo sequences using three-dimensional Fourier
transformation gradient-echo technique with gradient spoilers, allowing partition size of 1.5mm.
FAMILY 2.
FAMILY 3. The proband, his affected brother, and their
mother had 1.5T MRI. The boys were studied with 5.0mm
slice thickness. Their mother was studied with 3.0 to 4.0mm
thickness coronal fast SE T2-weighted and T1-weighted GE
scans, axial 5.00mm T2 and FLAIR images.
The proband, his mother, and maternal aunt
were studied using a three-dimensional T1 GE sequence giving approximately 170 slices with an isotropic voxel size of
1mm3. They also had 5mm thick axial proton-density, axial
and coronal T2-weighted and coronal FLAIR images.
FAMILY 4.
Chromosome and Mutation Analysis
High resolution chromosome analysis was performed in patients 1-IV:2, 1-IV:3, 1-IV:5, 1-IV:7, 1-IV:8, 2-III:2, 3-III:2,
and 4-III:4. The six coding exons of DCX were amplified by
polymerase chain reaction (PCR) using standard protocols6
and purified using the QIAquick PCR purification kit (Qiagen, Chatsworth, CA) and cycle sequenced on both strands
(BigDye Terminator chemistry; Applied Biosystems, Foster
City, CA).
We analyzed the sequence of the mutated DCX protein
using the PHD program,13 which predicts the secondary
structure and solvent accessibility of the protein from multiple sequence alignments. We also analyzed the sequence of
both DCX domains using the Conserved Domain Database
(http://www.ncbi.nlm.nih.gov:80/structure/cdd/cdd.shtml).
X-Inactivation Studies
Lymphocyte-derived DNA from patients 1-III:4, 1-IV:2,
1-IV:5, 1-IV:6, 2-I:2, 2-II:1, 2-II:2, 3-I:1, 3-II:1, 4-II:12,
and 4-II:16 and from eight control women was analyzed for
X-chromosome inactivation status. We performed the
HUMARA assay as previously described,12,14 with some
modifications. The products were analyzed on an ABI
PRISM 310 genetic sequencer (GENESCAN software; Applied Biosystems). X-inactivation was classified as random
(ratio 50:50 to ⬍80:20) or skewed (ratio ⱖ80:20).
Segregation of Highly Polymorphic Markers in the
Xq11.1-q22.3 Region
We analyzed 15 markers (DXS991, DXS1194, DXS1216,
DXS983, DXS8079, DXS1002, DXS1217, DXS990,
DXS8077, DXS1106, DXS1210, DXS1059, DXS1220,
DXS8081, DXS1001) in patients 2-I:2, 2-II:1, 2-II:2,
2-III:2, 3-I:1, 3-I:2, 3-II:1, 3-III:2. PCR amplifications were
performed, using the forward primers 5⬘ labeled with FAM
or HEX. PCR products were analyzed on ABI PRISM 310
sequencer (GENESCAN software; Applied Biosystems).
Mosaicism Analysis
We tested somatic mosaicism11,15,16 in patients 2-II:2 and
3-II:1, who had de novo mutations. Mosaicism was considered unlikely in carrier female subjects in Families 1 (1-IV:5)
and 4 (4-II:12 and 4-II:16).
The 166C3 G mutation in patients 3-II:1 caused the loss
of an RsaI restriction site. In this subject, we initially performed a fluorescent PCR reaction for both exons carrying
the mutation, at three different cycling conditions (25, 30,
35 cycles), followed by restriction enzyme digestion. We
used fluorescent (FAM) forward primers of exon 4.9 The
PCR and restriction enzyme–digested fragments were analyzed on ABI PRISM 310 sequencer, and mutant versus
wild-type peaks were quantitated by GENESCAN software.
Subsequently, each genomic PCR was repeated, digested by
specific restriction enzymes, and analyzed on 3% agarose gel.
Mutant versus wild-type band intensity was quantitated using NIH image software (http://rsb.info.nih.gov/nih-image/).
In patient 2-II:2, these methods were not applicable because
the 226C3 A mutation did not modify a restriction site.
Therefore, we analyzed only the sequence electropherograms
and single-strand conformation polymorphism bands.
Neuropsychological Testing
FAMILY 1. We studied three patients (1-III:4, 1-IV:2, and
1-IV:3) with the Wechsler Adult intelligence scale (WAISIII), one (1-IV:5) with the Wechsler intelligence scale for
children (WISC-III), and two younger boys (1-IV:7 and 1IV:8) with the Vineland Adaptive Behavior Scale.17 The
Guerrini et al: DCX Mutations
31
WISC-III also was administered to patients 1-IV:4 and
1-IV:6.
FAMILY 2. The proband was assessed using both the mental
and motor development indexes of the Bayley scales,18 and
his mother (2-II:2) was assessed using the WAIS-III.
The probands’ mothers in these families were assessed using the WAIS-III. Subject 4-II:12 was
judged using adaptive-behavior criteria.19
FAMILIES 3 AND 4.
Results
Clinical data are summarized in the Table and in
Fig 1.
Magnetic Resonance Imaging
FAMILY 1. The three affected boys had frontally predominant pachygyria (Fig 2A–C; cortical thickness,
15mm; normal, 3– 4mm) and a cell-sparse zone, consisting of a thin band of white matter in its superficial
part. In the two younger boys (1-IV:7 and 1-IV:8), a
thick cell-sparse SBH was visible in the parietooccipital
and inferior temporal lobes. The affected mother and
one of her daughters (1-IV:2) had thin SBH lying beneath the cortex of the superior, middle, and inferior
frontal gyri (see Fig 2D and E). Subject 1-IV:5, carrying the mutation, and her healthy brother (1-IV:4) had
normal MRI.
The proband had frontal pachygyria (cortical thickness, 15mm) and a band of white matter
within the anterior frontal cortex. The frontal lobes
were reduced in volume (see Fig 2G). FSPGR images
showed a thin parietooccipital SBH. The proband’s
mother had normal MRI (see Fig 2H).
FAMILY 2.
FAMILY 3. The proband and his affected brother (see
Fig 2K and J) had anteriorly predominant pachygyria
(cortical thickness, 15–20mm). Their mother had a
normal MRI (see Fig 2I).
The proband had mild frontal pachygyria
with underlying thin SBH (see Fig 2N). His mother
(see Fig 2M) and a maternal aunt (see Fig 2L) had a
mild reduction in the size of the frontal lobes.
FAMILY 4.
Neuropsychological Testing
Estimates of cognitive level are shown in the Table. In
Family 1, the two unaffected sibs had normal IQ
scores.
Chromosome and Mutation Analysis
Chromosome analysis was normal in all subjects studied. Sequence analysis of DCX 3,5,6,9 (AF034634) gave
the following results (see Fig 1).
Fig 1. Pedigrees of Families 1 to 4. Symbol identification is indicated in the box.
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Table. Main Clinical Features in 14 Subjects with DCX Missense Mutations from Four Families
Patients/Gender/Age (yr)
Age at Seizure Onset/
Type/Severity
Cognitive
Impairment
IQ Score
(FSIQ/VIQ/PIQ)/
Age Equivalents
(yr.mo)
Microcephaly
Brain MRI
⫹
No
⫹
SBH
SBH
Pachygyria ⫹
SBH
Normal
Pachygyria ⫹
SBH
Pachygyria ⫹
SBH
No seizure
No seizure
13yr/CPS/Resistant
Mild
Mild
Moderate
69/72/72a
74/72/79a
40/46/46b
No seizure
1 yr/AA ⫹ GTC/Resistant
Mild
Severe
No seizure
Severe
70/74/73b
1 yr 7 mos
2 yr 8 mosc
1 yr 8 mos
1 yr 9 mosc
No
⫹
Normal
Pachygyria ⫹
SBH
No seizure
3 mos/GCl/resistant
Mild/moderate
Severe
48/51/53a
0.3 mosd
FAM 3
3-III-1/F/32
3-III-1/M/1
3-III-2/M/3
FAM 4
4-II-12/F/55
No
⫹
⫹
Normal
Pachygyria
Pachygyria
No seizure
4 mo/GCl/resistant
8 mo/GCl/resistant
Mild
Severe
Severe
71/70/87a
N/A
N/A
No
N/A
No
15 yr/CPS ⫹ GTC/resistant
16 yr/GTC/controlled
Mild
4-II-16/F/51
Mild
59/55/68a
4-III-4/M/32
No
Small frontal
lobes
Small frontal
lobes
SBH
5 yr/GTC ⫹ CPS ⫹ DA/
resistant
Mild
N/A
FAM 1
I-III-4/F/46
I-IV-2/F/17
I-IV-3/M/16
I-IV-5/F/10
I-IV-7/M/6
No
⫹
I-IV-8/M/5
⫹
FAM 2
2-II-2/F/27
2-III-2/M/17mos
a
Wechsler Adult Intelligence Scale–III.
Wechsler Intelligence Scale for Children–III.
Vineland Adaptive Behavioural Scales.
d
Bayler Scale age equivalent.
b
c
MRI ⫽ magnetic resonance imaging. AA ⫽ atypical absence; CPS ⫽ complex partial seizures; DA ⫽ drop attack; FSIQ ⫽ full-scale IQ;
GCl ⫽ generalized clonic seizures; GTCS ⫽ generalized tonic-clonic seizure; N/A ⫽ not available; PIQ ⫽ performance IQ; SBH ⫽
subcortical-band heterotopia; VIQ ⫽ verbal IQ.
FAMILY 1. Patient 1-III:4 harbored a 412T3 C
change, resulting in a tyr138his (Y138H) substitution.
The same change was found in five additional subjects.
FAMILY 2. Patient 2-III:2 and his mother harbored a
226C3 A change, resulting in an arg76ser (R76S) substitution.
Patient 3-III:2 and his mother harbored a
166C3 G change, resulting in an arg56gly (R56G)
substitution.
FAMILY 3.
FAMILY 4. Patient 4-III:4, his mother, and an aunt
with epilepsy (4-II:12) harbored a 532C3 T change,
resulting in an arg178cys (R178C) substitution.
None of the amino acid changes observed in these
families appeared to be a polymorphism, because they
were not found in more than 150 DCX alleles tested in
our laboratory.
The alignment results for DCX obtained using the
Conserved Domain Database showed that in the con-
sensus sequence of the DCX domain (smart00537.4)
(http://smart.embl-heidelberg.de) the two arginine residues at amino acids 56 and 76 are highly conserved.
They may play an important role in the DCX domain
structure, compared with the arginine at amino acid
178.
X-Inactivation Studies
X-inactivation studies in Families 1 and 4 showed a
normal pattern in the five affected female subjects
tested. An unaffected girl in Family 1 (1-IV:6) and
normal controls also had a normal pattern. Families 2
and 3 showed significant X-inactivation skewing in carrier mothers with a 90:10 ratio. In both mothers, the
preferentially inactivated allele was not the one inherited by their affected sons. However, the HUMARA
and DCX genes are located approximately 35cM apart,
implying a high chance of recombination. In Family 2,
genotyping of relevant patients (I:2, II:1, II:2, and
III:2) in the Xq11.1-22.3 region did not allow identification of the number and localization of putative
Guerrini et al: DCX Mutations
33
Fig 2. (A–F) Brain magnetic resonance imaging (MRI) scans of subjects carrying the 412T3 C DCX mutation in Family 1 (axial
sections; T2W; 1.5T for all, except E; 1T). Subjects 1-IV:3 (A), 1-IV:7 (B), and 1-IV:8 (C) are boys. Subjects’ ages at the time of
scanning were 15 years (A), 10 months (B), and 12 months (C). MRI shows thin anterior subcortical band heterotopia (arrows).
All three boys have an anterior thin band. Subjects 1-IV:7 (B) and 1-IV:8 (C) in addition have a thick posterior band. Subjects
1-III:4 (D), 1-IV:2 (E), and 1-IV:5 (F) are female. An axial section and the coronal section conducted through the axis of the
white line are shown for each subject. A very thin band of subcortical heterotopia is visible in the frontal lobes in Subjects 1-III:4
(D) and 1-IV:2 (E). Subject 1-III:4 (D) has in addition mild frontal pachygyria. No abnormality is visible in Subject 1-IV:5 (F).
(G–H) Brain MRI (FSPGR; 1.5T) of Subjects 2-III:2 (G) and 2-II:2 (H) from Family 2, both carrying a 226C3 A mutation of
DCX. MRI of Subject 2-III:2 (G, boy) shows severe pachygyria in the frontal lobes (upward pointing arrows) and subcortical
band heterotopia in the parietal lobes (downward pointing arrows). MRI scan of his mother, 2-II:2 (H), is normal. (I–K) Brain
MRI scans (1.5T) of Subjects 3-II:1 (I; T2 weighted), 3-III:1 (J; proton density), and 3-III:2 (K; inversion recovery) in Family 3.
A 166C3 G mutation of DCX was demonstrated in the mother (I) and in the living proband (K). The brain MRI of both boys
(J, K) shows severe pachygyria. MRI scan of the mother (I) is normal. (L–N) Brain MRI (1.5T; T1) of Subjects 4-II:12 (L),
4-II:16 (M), and 4-III:4 (N) carrying a 532C3 T mutation of DCX in Family 4. The proband (N) has a very thin linear (bilateral) layer of heterotopic gray matter underlying the frontal cortex (arrow). His mother (M) and maternal aunt (L), both having
epilepsy, have normal scans.
crossover. In contrast in Family 3, segregation analysis
of markers in patients 3-I:1, 3-I:2, 3-II:1, and 3-III:2
showed a single crossover in the affected boy (3-III:2),
34
Annals of Neurology
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between DXS1217 and DXS8077, proximally to DCX
(data not shown). Probands’ maternal grandmothers
(2-I:2 and 3-I:1) showed a normal inactivation pattern.
Mosaicism Detection
In patient 3-II:1, the area of the GENESCAN peaks
representing the mutant and wild-type alleles was comparable. Agarose gel quantification of the mutant and
wild-type bands showed the same intensity. These results exclude somatic mosaicism in blood lymphocytes.
In Subject 2-II:2, sequence electropherograms and
single-strand conformation polymorphism analysis did
not suggest mosaicism. Reliability of this method is
limited, however.
Discussion
Doublecortin
The DCX gene codes for a 360 amino acid, 46kD protein, which is expressed in migrating neurons and the
cortical plate during embryonic development.2,3,20,21 A
highly conserved region is present in two repeats (pep1
or R1: amino acids 51–135 and pep2 or R2: amino
acids 178 –259) in the N-terminal portion.11,22 These
conserved domains are essential for microtubule polymerization and bundling, which are crucial for correct
neuronal migration. R1 binds tubulin, enhances tubulin assembly, and plays a role as homodimerization domain of DCX and as LIS1–DCX interaction domain.23 Mutations in the R1 and R2 domains result in
defective tubulin interaction in vitro.22,24,25 Although
some missense mutations retain residual polymerizing
activity, others produce severely defective interactions.
The first 46 amino acids and the inter-repeat region
are not themselves sufficient for coassembly with microtubules and probably act as regulators of DCXmicrotubule binding.25
Genotype-phenotype studies indicate that missense
mutations tend to cluster in the R1 and R2 repeats12
and support experimental data on the functional importance of the N-terminal region.
Mutations in Families 1 to 4
The mutations observed in these four families produced mild clinical consequences, including five female
carriers with normal brain MRI, mild cognitive impairment, and epilepsy in two. Cognitive impairment and
epilepsy, without detectable structural abnormality, can
result from neuronal heterotopia below the resolution
of MRI. Alternatively, a mildly abnormal DCX function, or a severe dysfunction affecting only a minority
of neurons, may affect cognition and neuronal excitability because of their molecular repercussions on microtubules and neuronal connectivity,21 independently
from any migration abnormality in the neocortex. For
example, Dcx mutant mice, both hemizygous and heterozygous, show subtle lamination defects in the hippocampus and learning deficits, in the absence of any
abnormality in neocortical lamination.26 A mild phenotype in female subjects, including normal MRI, was
common to all four families. Although in Families 1
and 4 the phenotype was mild in both genders, in
Families 2 and 3 affected boys had severe phenotypes.
Because the severity of pachygyria-SBH can be roughly
correlated with that of the DCX mutations identified,3
it is likely that the normal MRI in carrier mothers in
Families 2 and 3 may be explained by additional factors, including favorable X-inactivation skewing or somatic mosaicism.
In Family 1, the tyr138his change falls in the hinge
region (amino acids 135–178), whose functional role is
poorly understood and where no missense mutations
had been detected previously. This mutation has mild
functional consequences that might result from abnormal folding in the inter-repeat region (PHD
prgram13). However, as the tyr138his change falls just
three amino acids after the end of pep1, it might adversely affect the function of pep1 rather than the
hinge. There was no altered pattern of X-inactivation
in affected female subjects in this family. Clinical presentations included three affected boys with anterior
mild pachygyria-SBH and moderate to severe mental
retardation, the affected mother and one daughter with
thin frontal SBH and mild mental retardation, and another daughter carrying the mutation having mild
mental retardation with normal imaging.
In Family 4, the arg178cys change falls in the initial
portion of the pep2 region, in the putative ␤-sheet
structure.25 Carrier female subjects had epilepsy and
mild mental retardation with normal brain MRI. The
proband had mild mental retardation, epilepsy, and a
thin frontal band, featuring the mildest phenotype that
was ever observed in a male subject carrying a DCX
mutation. An arg178leu change previously had been
reported in a pedigree in which female subjects had
manifest SBH and high miscarriage rate suggesting a
male lethal phenotype.3 Therefore, these different
amino acid changes at residue 178 alter the DCX function with variable severity.
Because no skewed X inactivation or somatic mosaicism appeared to operate in lymphocyte DNA of affected female subjects in Families 1 and 4, the phenotype probably correlated with the mild mutations.
Families 2 and 3 harbored two novel missense mutations (arg76ser and arg56gly) within R1, which is involved in tubulin binding and microtubule polymerization. The DCX repeats may take the form of a ␤-grasp
superfold containing a ␤2␣␤3 architecture.25 The arginine at position 56 is located in the first ␤-sheet of
the first DCX domain, whereas the arginine at position
76 is proximal to the ␣-helix structure. Considering
the structural predictions of the DCX repeats,25 such
mutations should affect protein folding, modifying
DCX activity. Analysis of the DCX domain structure
indicates that the arginine residues at positions 56
(Family 3) and 76 (Family 2) are more conserved than
Guerrini et al: DCX Mutations
35
the arginine at position 178 (Family 4). Although
pachygyria in affected boys in Families 2 and 3 is not
surprising, a normal brain MRI in their mothers is unexpected.
retardation31,32 also including RSK2,33,34 ATR-X,35
MECP2,36 and ARX.37,38
References
Possible Role of Nonrandom X-Inactivation in
Phenotypic Heterogeneity
The skewed X-inactivation observed in the blood lymphocytes of carrier female subjects in Families 2 and 3
could indicate that preferential inactivation of the mutant allele may explain their mild phenotype.3 Although nonrandom X-inactivation was not observed
previously with DCX mutations,12,27 only female subjects with SBH had been tested. X-inactivation ratios
can be assumed to have a distribution that follows a
bell-shaped curve in the female population.28 Most carrier female subjects have a balanced inactivation of the
normal and mutated allele. However, a minority of
subjects (at the extremes of the curve) have a proportion of inactivation that enhances the phenotypic variability,29 including subjects in whom preferential inactivation of the mutated allele causes mild
nonsyndromic phenotypes with consequent under ascertainment.
Although skewing of X-inactivation is a plausible
mechanism of phenotypic heterogeneity, it ultimately
cannot be demonstrated, because rates in blood lymphocytes might not reflect those in the brain.
Somatic mosaicism has been associated with enhanced phenotypic heterogeneity in SBH.11,15,16 We
did not observe mosaicism in blood lymphocytes of
two female subjects harboring de novo mutations (2II:2 and 3-II:1). Although the ratio of mosaicism in
the brain is better explored by hair root analysis (ectodermal derivatives),15 previously reported somatic DCX
mutations were detectable in blood lymphocytes.11,15,16
Clinical Implications
The carrier status of female subjects with DCX mutations causing nonsyndromic mental retardation, with
or without epilepsy, is easily under recognized. DCX
mutations would not have been looked for in these
families, but for the birth of the severely affected boys.
The risk for recurrence of either SBH or XLIS after
the birth of a first child was considered to be low if the
mother had normal MRI.30 However, the possibility of
carrier female subjects with nonsyndromic mild mental
retardation, and of unaffected female subjects with somatic and germline mosaicism,11 makes mutation analysis in the mothers of an affected child important, even
when brain MRI is normal. The limited reproductive
disadvantage accompanying mild mutations enhances
the relevance of this observation.
DCX is to be considered among the X-linked genes
that cause syndromic as well as nonsyndromic mental
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1. Reiner O, Carrozzo R, Shen Y, et al. Isolation of a MillerDieker lissencephaly gene containing G protein beta-subunitlike repeats. Nature 1993;364:717–721.
2. Des Portes V, Pinard JM, Billuart P, et al. A novel CNS gene
required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome. Cell
1998;92:51– 61.
3. Gleeson JG, Allen KM, Fox JW, et al. Doublecortin, a brainspecific gene mutated in human X-linked lissencephaly and
double cortex syndrome, encodes a putative signaling protein.
Cell 1998;92:63–72.
4. Sossey-Alaoui K, Hartung AJ, Guerrini R, et al. Human doublecortin (DCX) and the homologous gene in mouse encode a
putative Ca2⫹-dependent signaling protein which is mutated
in human X-linked neuronal migration defects. Hum Mol
Genet 1998;7:1327–1332.
5. Pilz DT, Kuc J, Matsumoto N, et al. Subcortical band heterotopia in rare affected males can be caused by missense mutations in DCX (XLIS) or LIS1. Hum Mol Genet 1999;8:
1757–1760.
6. Pilz DT, Matsumoto N, Minnerath S, et al. LIS1 and XLIS
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