doi:10.1093/brain/awl125Brain (2006) Page 1 of 15
Periventricular heterotopia: phenotypic
heterogeneity and correlation with
Filamin A mutations
E. Parrini,1,* A. Ramazzotti,1,* W. B. Dobyns,12D. Mei,1F. Moro,1P. Veggiotti,3C. Marini,1
E. H. Brilstra,16B. Dalla Bernardina,4L. Goodwin,17A. Bodell,9M. C. Jones,13M. Nangeroni,5
S. Palmeri,6E. Said,18J. W. Sander,14P. Striano,7Y. Takahashi,19L. Van Maldergem,20G. Leonardi,8
M. Wright,15C. A. Walsh10,11and R. Guerrini1,2
1Research Institute I.R.C.C.S. Stella Maris Foundation and2Department of Child Neurology and Psychiatry, University of
Pisa,3Child Neuropsychiatry Department, Neurological Institute Casimiro Mondino Foundation I.R.C.C.S., University of
Pavia,4Department of Pediatrics and Child Neuropsychiatry, Verona University Medical School,5E. Agnelli Hospital,
Pinerolo, Torino,6Department of Neurological and Behavioural Sciences, University of Siena,7Department of Neurological
Sciences, University of Napoli Federico II,8Unit of Child Neurology and Psychiatry Fatebenefratelli Hospital, Milano, Italy,
9Walsh Laboratory, Harvard Medical School,10Division of Neurogenetics and Howard Hughes Medical Institute,
Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School,11Program in Biological and
Biomedical Sciences, Harvard Medical School, Boston, MD,12Department of Human Genetics, Neurology and Pediatrics,
University of Chicago, IL,13Children Hospital and Health Centre, San Diego, CA, USA,14Department of Clinical and
Experimental Epilepsy, Institute of Neurology, London,15Institute of Human Genetics International Centre for Life,
Newcastle-upon-Tyne, UK,16Department of Medical Genetics, University Medical Center, Utrecht, The Netherlands,
17Department of Genetics, Nepean Hospital, Penrith, Australia,18St Luke’s Hospital, Gwardamangia, Malta,19National
Epilepsy Centre, Shizuoka Medical Institute of Neurological Disorders, Shizuoka, Japan and20Human Genetics Centre,
Institute of Pathology and Genetics-Loverval, Belgium
*These authors contributed equally to this work.
Correspondence to: Prof. Renzo Guerrini, Division of Child Neurology and Psychiatry I.R.C.C.S. Stella Maris
Foundation, University of Pisa-via dei Giacinti, 2-56018 Calambrone, Pisa, Italy E-mail: email@example.com
Periventricular heterotopia (PH) occurs when collections of neurons lay along the lateral ventricles or just
nodular heterotopia (PNH), featuring contiguous heterotopic nodules, mega cisterna magna, cardiovascular
malformations and epilepsy. FLNA encodes an F-actin-binding cytoplasmic phosphoprotein and is involved in
early brain neurogenesis and neuronal migration. A rare, recessive form of bilateral PNH with microcephaly
and severe delay is associated with mutations of the ADP-ribosylation factor guanine nucleotide-exchange factor-2
(ARFGEF2) gene, required for vesicle and membrane trafficking from the trans-Golgi. However, PH is a hetero-
geneous disorder.We studied clinical and brain MRI of 182 patients with PH and, based on its anatomic dis-
tribution and associated birth defects, identified 15 subtypes. Classical bilateral PNH represented the largest
group (98 patients: 54%). The 14 additional phenotypes (84 patients: 46%) included PNH with Ehlers–Danlos
fronto-perisylvian or temporo-occipital polymicrogyria, posterior PNH with hydrocephalus, PNH with micro-
cephaly, PNH with frontonasal dysplasia, PNH with limb abnormalities, PNH with fragile-X syndrome, PNH
with ambiguous genitalia, micronodular PH, unilateral PNH, laminar ribbon-like and linear PH. We performed
mutation analysis of FLNA in 120 patients, of whom 72 (60%) had classical bilateral PNH and 48 (40%) other PH
phenotypes, and identified 25 mutations in 40 individuals. Sixteen mutations had not been reported previously.
Mutations were found in 35 patients with classical bilateral PNH, in three with PNH with EDS and in two with
unilateral PNH. Twenty one mutations were nonsense and frame-shift and four missense. The high prevalence
of mutations causing protein truncations confirms that loss of function is the major cause of the disorder. FLNA
mutations were found in 100% of familial cases with X-linked PNH (10 families: 8 with classical bilateral PNH,
#The Author (2006). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: firstname.lastname@example.org
Brain Advance Access published May 9, 2006
1 with EDS and 1 with unilateral PH) and in 26% of sporadic patients with classical bilateral PNH. Overall,
mutations occurred in 49% of individuals with classical bilateral PNH irrespective of their being familial or
sporadic. However, the chances of finding a mutation were exceedingly gender biased with 93% of mutations
occurring in females and 7% in males. The probability of findingFLNA mutations in other phenotypes was 4% but
was limited to the minor variants of PNH with EDS and unilateral PNH. Statistical analysis considering all
42 mutations described so far identifies a hotspot region for PNH in the actin-binding domain (P < 0.05).
Keywords: periventricular heterotopia; filamin A; FLNA; mutation; genetic counselling
Abbreviations: CH = calponin homology; DHPLC = denaturing high performance liquid chromatography;
EDS = Ehlers–Danlos syndrome; NMD = nonsense-mediated mRNA decay; OPD = otopalatodigital; PH = periventricular
heterotopia; PMG = polymicrogyria; PNH = periventricular nodular heterotopia; RT–PCR = reverse
transcription–polymerase chain reaction
Received January 18, 2006. Revised April 1, 2006. Accepted April 10, 2006
Defects of the human Filamin A gene (FLNA; chromosomal
locus Xq28) cause X-linked periventricular nodular hetero-
topia (PNH; OMIM 300049) (Eksioglu et al., 1996; Fox et al.,
1998), a malformation of neuronal migration (Barkovich
et al., 1992) characterized by nodules of neurons in an
inappropriate location adjacent to the walls of the lateral
ventricles, the location of the embryonic ventricular zone
(Dobyns et al., 1996). PNH can be bilateral or unilateral.
Periventricular heterotopia (PH) may also be laminar, rather
than nodular. Most patients with PH have seizures whose
severity and age at onset are variable (Huttenlocher et al.,
1994; Guerrini and Carrozzo, 2001).
PNH may also be observed in patients with chromo-
somal rearrangements, such as duplication of 5p15.1 or
5p15.33 (Sheen et al., 2003b), or with mutations of
ARFGEF2, which cause a rare autosomal recessive syndrome
of PNH and microcephaly (Sheen et al., 2004a). PNH has
also been associated with hydrocephalus (Sheen et al.,
2004b), frontonasal malformation (Guerrini and Dobyns,
1998), severe mental retardation and syndactyly (Dobyns
et al., 1997; Fink et al., 1997), Ehlers–Danlos syndrome
(EDS) (Thomas et al., 1996; Sheen et al., 2005) or nephrosis
(Palm et al., 1986). Whether each of these variants represents
a truly distinct compound disorder with a specific genetic
mechanism remains to be determined.
FLNA codes for the F-actin-binding cytoplasmic cross-
linking phosphoprotein filamin A (FLNA) (Carroll et al.,
1982; Chen et al., 1989), composed of three major func-
tional domains: an actin binding domain (ABD), at the
N-terminus, consisting of tandem pairs of calponin homol-
ogy domains (CH1 and CH2), a rod domain consisting of
23 immunoglobulin-like repeats interrupted by two hinge
regions (‘hinge 1’ and ‘hinge 2’) and a C-terminal domain,
which is important for dimerization and binding to several
membrane receptors (Noegel et al., 1989; Gorlin et al., 1990;
Hock et al., 1990).
The specific roles of filamin A and its association with
pathological conditions are still to be fully understood.
Additional functional complexity is conferred by the exis-
tence and by the time/tissue-specific expression of the two
human paralogous genes Filamin B and Filamin C (Stossel
et al., 2001; van der Flier and Sonnenberg, 2001). The
Y-shaped homodimerized FLNA promotes the organization
of actin filaments in orthogonal networks of the cytoskele-
ton. A similar mechanism might also be hypothesized for
filamin B and C, even if their heterodimerization has been
demonstrated only in vitro (Himmel et al., 2003). In addi-
tion, filamin A and filamin B co-localize within neuronal
precursors and heterodimerization of these two isoforms
has been hypothesized (Sheen et al., 2002). Interactions of
the dimer with integrin receptors, transmembrane proteins
and several signalling proteins are believed to integrate
extracellular and cytoplasmic signals with cellular cytoskele-
ton rearrangements and membrane reshaping (Meyer et al.,
1997; Loo et al., 1998; Ott et al., 1998; Dulabon et al., 2000;
Tu et al., 2003). Filamin homologues have been shown to be
implicated in regulation of cell stability, protrusion and
motility across various biological systems (Stendahl et al.,
1980; Cunningham, 1995; Ott et al., 1998). The mouse
Filamin A orthologue was abundantly expressed in the cell
soma and leading processes of migratory neurons and is
widely distributed across the cortical mantle, reaching
the highest levels in the ventricular zone within the cortex
during neurogenesis (Sheen et al., 2002). FLNA likely influ-
ences neuroblast migration during cortical development
in vertebrates, and PH in humans likely results from disrup-
tion of this process (Sheen et al., 2001).
Mutations of the FLNA gene have also been associated
with the otopalatodigital (OPD) syndrome spectrum,
which includes OPD I (OPD1; OMIM 311300) and OPD
II (OPD2; OMIM 304120), frontometaphyseal dysplasia
(FMD; OMIM 305620) and Melnick-Needles syndrome
(MNS; OMIM 309350) (Robertson et al., 2003).
Since 1998, 45 mutations of FLNA have been reported
in patients with PNH or the OPD syndrome spectrum
(Robertson et al., 2003; Hidalgo-Bravo et al., 2005;
Page 2 of 15Brain (2006) E. Parrini et al.
Stefanova et al., 2005), including 25 missense, 3 nonsense
and 8 splice site mutations, plus 7 deletions and 2 insertions
(mutations related to PNH are summarized in Table A as
Supplementary online material). To extend these observa-
tions, we have reviewed clinical and brain imaging data in
182 individuals with PH, and identified 25 FLNA mutations
including 16 novel mutations in 40 of the 120 patients
tested. We report genotype–phenotype analysis in these
40 patients, and describe 15 clinically distinct subgroups
including 3 associated with FLNA mutations in many but
not all patients, 4 previously reported syndromes (Guerrini
and Dobyns, 1998; Sheen et al., 2004a; Wieck et al., 2005),
and 8 novel phenotypes.
Patients and methods
Clinical information, brain MRI and
Clinical information and brain MRI of 182 patients with PH were
either collected at the Division of Child Neurology and Psychiatry,
University of Pisa, or obtained from the patients’ referring hospitals.
Informed consents were obtained from the respective human
subject institutional review boards. We collected clinical informa-
tion and assessed brain MRI for the purpose of classifying patients.
We analysed the shape, location and symmetry of PH; the presence
of any associated brain malformations or other congenital anoma-
lies; and specific syndrome diagnosis (Table 1).
Seventeen patients were included in the ‘unclassified’ group
because either neuroimaging was just sufficient to detect PH, but
not clear enough to fully characterized its extent and morphology,
or clinical information was insufficient for delineating a syndrome
Once the phenotype characterization was obtained using the
criteria described above, we performed mutation analysis of
the FLNA gene in subsets of patients belonging to most of the
phenotypic subclasses and analysed the rate of mutations according
to specific phenotype, familial or sporadic occurrence and gender.
Genomic DNA samples
Genomic DNA of 120 individuals with PH (64 women and 56 men)
was extracted from peripheral blood leucocytes using a DNA
isolation kit (DNAzol, MRC, Cincinnati, OH, USA).
Molecular analysis of FLNA
We carried out mutation screening of the coding regions of FLNA
(Gen Bank accession number NM_ 002834) by denaturing high
performance liquid chromatography (DHPLC), according to the
manufacturer’s specifications (Transgenomics, La Jolla, CA, USA).
The 47 exons covering coding regions and their respective upstream
and downstream intron–exon boundaries were amplified by PCR.
Primer sequences, PCR conditions and DHPLC analysis tempera-
tures are available on request. Amplicons from male subjects were
mixed with half volume of PCR product of the same FLNA region
amplified by the same conditions from an unaffected female DNA.
PCR products that showed an alteration in the DHPLC elution
profile were purified using the GenElute PCR clean-up kit
(Sigma Aldrich, Italy) and cycle sequenced on both strands using
the BigDye Terminator v1.1 chemistry (Applied Biosystems, Foster
City, CA, USA) and an ABI3100-Avant automated sequencer
(Applied Biosystems). The nucleotide changes observed (see
Results) were not found in 125 DNA samples of mixed ethnic
For Patient 2a, who carries a silent mutation in exon 31
(Table 2), we used the Berkeley Drosophila Genome Project
(BDGP) splice database (http://www.fruitfly.org/seq_tools/splice.
html) to predict in silico whether the nucleotide change would
alter the mRNA splicing of exon 31, its neighbouring exons, or
both. Total RNA was isolated from a lymphoblastoid cell line
using the GenElute mammalian total RNA kit (Sigma Aldrich,
Italy). Reverse transcription (RT) was performed using 1mg total
RNA with random hexanucleotide primers and Im ProM-II Reverse
Transcriptase (Promega, Italy). The cDNA was amplified by the
following primer pair: F31-RT-F (50-ctgtggacactaaggcggc-30)–F32-
RT-R (50-gctgctgagaccgtagagg-30); the RT product was then
sequenced as described above. In order to assess nonsense-
mediated mRNA decay (NMD), RT–PCR was performed on the
patient’s lymphoblastoid cell line treated for 24 h with 100 mg/ml
cycloheximide, an NMD inhibitor, as previously described (Usuki
et al., 2004). RT–PCR products were cloned into the pGEM-T-easy
vector (Promega, Italy) according to the manufacturer’s protocol.
The resulting colonies were amplified with primers (T7 and SP6)
flanking the vector’s poly-cloning site and verified for the presence
of inserted fragments by gel electrophoresis.
In the same patientwe
X-chromosome inactivation with the androgen receptor (AR)
assay, using a fluorescence-labelled primer on leucocyte-derived
DNA (Allen et al., 1992). The PCR products were analysed on
an ABI PRISM 3100 Avant genetic sequencer, and the allele sizes
were determined by GENEMAPPER software (Applied Biosystems).
Patient 6a carried a substitution of 2 nt in exon 25 (Table 2).
To verify whether such changes were in cis or in a trans configura-
tion, we subcloned the PCR product of exon 25 obtained from
genomic DNA into the pGEM-T-easy vector (Promega, Italy)
and subsequently sequenced the resulting insert using T7 and
also testedthe patternof
To establish whether FLNA contains mutational hotspots, we
performed statistical analysis considering all 42 mutations described
so far, including the 16 new mutations reported herein. Mutations
were tabulated according to their location in the gene, considering
the coding exons plus the 10 nt upstream and downstream from
each exon: ABD (904 nt), Rod domain 1 + Hinge 1 region (5029 nt),
Rod domain 2 + Hinge 2 (2629 nt) and C-terminal domain (299
nt). For comparison, Fisher’s exact test was applied to verify the
hypothesis of absence of hotspot regions for FLNA mutations, tak-
ing into account the relative sizes of the four domains. Fisher’s exact
test is a method of performing inference in two-way contingency
tables using exact distributions.
We applied the x2test with one degree of freedom to establish if
significant skewing of the sex ratio occurred in patients with
classical bilateral PNH but no mutations of the FLNA gene.
Based on the comparison of clinical and brain MRI findings
of the 182 patients, we defined 15 phenotypic subclasses
Anatomoclinical spectrum of PH and FLNA mutations Brain (2006)Page 3 of 15
Table 1 PH classes, patients and FLNA analysis
1.1. Bilateral diffuse
1.1.1 With frontonasal dysplasia
1.2. Bilateral diffuse sparing
1.2.1. ‘Classical’ bilateral PNH
1.2.2. With Ehlers–Danlos syndrome
1.2.3. With micronodules
1.2.4. With ambiguous genitalia
1.2.5. With limb abnormalities
1.2.6. With microcephaly
1.3. Bilateral temporo-occipital
1.3.1. With polymicrogyria
1.3.2. With cerebellar hypoplasia
1.3.3. With hydrocephalus
1.4. Bilateral anterior,
frontal ! to trigones
1.5. Unilateral diffuse
sparing temporal horns
2.1. Diffuse linear
2.2. Posterior ribbon-like
*A proband had BPNH associated with EDS, however, her mother had ‘classical’ BPNH.
Page 4 of 15Brain (2006) E. Parrini et al.
of PH whose anatomical characteristics are summarized in
Table 1 and Figs 1–4. Ninety-eight patients (54%) had
classical bilateral PNH. Of the remaining 84 (46%) patients,
26 (31%) had bilateral PNH associated with other brain
malformations, 19 (23%) had bilateral PNH associated
with non-neurological abnormalities and 22 (26%) had
either unilateral PNH, micronodules or laminar heterotopia.
In most phenotypic subclasses the morphological character-
istics of PH appeared to be sufficiently specific. The remain-
ing 17 (20%) had different forms of PNH that did not fit
features of any specific subclass.
Classical bilateral PNH
Ninety-eight patients (54 females, 44 males; age range: 2–
64 years; mean age: 20 years) had bilateral symmetric nodules
of grey matter lining the lateral ventricles, especially the
frontal horns and ventricular bodies, with limited extension
in the occipital horns, almost always sparing the temporal
horns (Fig. 1A–E), or with minor temporal involvement,
which, however, was never accompanied by abnormal
hippocampal morphology. In 22 patients (8 probands)
(22.4%) the disorder was familial, always with a pattern
suggesting X-linked inheritance.
Most patients in this group had normal intelligence or
mild mental retardation. Three patients, none having
FLNA mutations, had moderate to severe mental retardation.
Epilepsy was observed in 73 patients (72%). Age at seizure
onset varied from the neonatal period to 43 years (mean
12 years). Several different seizure types were observed,
with most patients having focal epilepsy. Seven patients
had early epileptic encephalopathies with infantile spasms
and tonic seizures. Seizures were well controlled or rare in
82% of those with epilepsy.
Mutations of the FLNA gene were observed in 35 (49%)
of the 72 patients who were tested, including 33 (77%)
of 43 females and 2 (7%) of 29 males. Almost all patients
with FLNA mutations had mild to moderate cerebellar
vermis hypoplasia, and many also had cardiovascular
abnormalities. In particular, twenty patients had either insuf-
ficiency of the aortic valve, patent ductus arteriosus (PDA)
or idiopathic thrombocytopenia, or an association of
them. Beside these abnormalities, no remarkable clinical
or anatomical differences were detected between patients
with or without FLNA mutations.
Bilateral posterior PNH (temporal horns,
trigones and occipital horns) with
hippocampal malformation and
Ten patients (7 females, 3 males; age range: 5–50 years; mean
age: 25 years) had bilateral PNH with nodules restricted to
the trigones and temporal and occipital horns. Heterotopia
surrounded the hippocampi that were under-rotated and
rounded (Fig. 2A and B). The number of patients is too
small to estimate gender ratio. All patients were sporadic
with no reported consanguinity. All 10 had severe cerebellar
vermis hypoplasia, and 8 had moderate to severe hypoplasia
of the cerebellar hemispheres as well (Fig. 2C–E). Two
Table 2 Clinical features of patients with PH and new FLNA mutations
Mutation Patient Gender
Sequence variationExon Protein Location in protein
IVS5 +2 T!A
IVS44 –2 A!G
IVS47 +8 A!G
c.[4437 A!G;4438 C!A]
c.[4437 A!G;4438 C!A]
c.[4437 A!G;4438 C!A]
c.[4437 A!G;4438 C!A]
4 repeat 1—rod 1
repeat 15—rod 1
repeat 15—rod 1
repeat 22—rod 2
repeat 4—rod 1
repeat 4—rod 1
repeat 12—rod 1
repeat 12—rod 1
repeat 12—rod 1
repeat 12—rod 1
repeat 13—rod 1
repeat 21—rod 2
repeat 11—rod 1
repeat 11—rod 1
repeat 15—rod 1
u = unknown.
Anatomoclinical spectrum of PH and FLNA mutationsBrain (2006) Page 5 of 15
patients had agenesis of the corpus callosum and four had
thinning of the corpus callosum. Cerebellar signs were
present in all, though their severity varied from a severe
cerebellar syndrome that initially prompted brain imaging
in most patients to mild dysmetria, nystagmus and dysar-
thria. Seven patients had epilepsy, which was always focal.
Age at seizure onset ranged from 1 to 33 years (mean age:
13 years). Five patients were seizure free or had occasional
seizures and the remaining 2 had uncontrolled seizures.
Cognitive level varied from normal to mildly impaired.
No mutations of the FLNA gene were observed in the
7 patients analysed.
Bilateral posterior PNH and
In two boys, mostly non-contiguous PNH lining the poster-
ior bodies, trigones and temporal and occipital horns were
associated with overlying polymicrogyria (PMG) involving
the temporal, parietal and occipital lobes. A series of 20 such
patients (15 males and 5 females) was reported in a compa-
nion paper (Wieck et al., 2005). All had developmental delay
and mental retardation, and most had epilepsy but the
severity was variable. Both patients in this paper (and all
in the companion paper) were sporadic with no reported
Fig. 2 Brain imaging in two patients with bilateral PNH involving the temporo-occipital horns and trigones with hippocampal malformation
and cerebellar hypoplasia. A–D are from the same patient, a 6-year-old, girl. A sagittal section through the bodies of the lateral ventricles
(A), shows that there is no subependymal heterotopia at this level; a lower sagittal section, through the temporal horns (B) shows
contiguous bilateral subependymal heterotopia (arrowheads) which reaches the tip of the temporal horns where it merges with the
hippocampal formations. Coronal sections show heterotopia surrounding the trigones (C) and merging with the hippocampal formation (D)
as well as severe cerebellar hypoplasia. A sagittal section in a 15-year-old, girl (E), shows severe cerebellar hypoplasia involving both
cerebellar hemispheres and the vermis (C and D).
Fig. 1 Brain imaging in five females with FLNA mutations demonstrates extensive contiguous (B, C, D) or non-contiguous (A) PNH
involving the body and trigones of the lateral ventricles with overlying normal cortex, except that heterotopia are seen only on the right in
one patient (B). The sagittal section (E) shows extensive heterotopia beneath the walls of the body and trigones of the lateral ventricle,
with sparing of the temporal horn and hippocampal formation. The associated FLNA mutations in these patients are: Q668X in
patient 2-II.2 (A), S149P in patient 2-III.1 (as reported in Guerrini et al., 2004) (B), c.4038 delG in patient 5-I.2 (C) who is the mother
of 5-II.2, A39G in patient F8 who has EDS (D), and IVS5+2T!A in Patient 1 (E).
Page 6 of 15 Brain (2006) E. Parrini et al.
Bilateral frontal-perisylvian PNH and
In seven patients (6 males, 1 female), small and mostly
non-contiguous nodules lining the frontal horns, bodies of
the lateral ventricles and the trigones were associated with
overlying frontal and perisylvian PMG, occasionally extend-
ing to the parietal cortex (Fig. 3A). All patients were sporadic
and no consanguinity was reported in their families. Severe
developmental delay, present in all, was accompanied in
most by early onset seizures. No FLNA mutations were
found in the four patients tested. Four of the patients in
this group were also included in the companion paper by
Wieck et al. (2005); three additional patients had not been
reported before. The clinical characteristics of our seven
patients are similar to those described in detail in Wieck’s
series. Combining the new patients included here and
those previously reported, the male to female rate is 9
to 2, which confirms a significant skewing of the sex ratio
(x2test: P = 0.034).
Bilateral posterior PNH with
Five patients (3 males, 2 females; age range: 3–35 years; mean
age: 11 years) had small non-contiguous nodules or small
clusters of nodules in the occipital and posterior temporal
horns and trigones in association with hydrocephalus
(Fig. 3B). In two unrelated patients, PNH but not hydro-
cephalus was present in other family members (Sheen et al.,
2004b; Family 2-III.1 and Family 3-II.3) who were not
included in this study. Another patient had Chiari malfor-
mation type 1 with caudally-displaced cerebellar tonsils,
syringomyelia and tethered cord. Four patients had severe
developmental delay, three had epilepsy, two had spastic
quadriparesis and one had PDA. Mutation analysis of
FLNA was negative in the three patients studied.
Bilateral PNH with microcephaly
Two siblings, a boy and a girl from a Turkish, consan-
guineous family, had bilateral diffuse PNH, sparing the
temporal horns, associated with microcephaly with head
circumference – 2 SD. These two patients were included
in the paper describing mutations of the ARFGEF2 gene
in recessive PNH with microcephaly (Family 2 in Sheen
et al., 2004a). Both children had severe developmental
delay, spastic quadriparesis and early-onset refractory infan-
tile spasms with hypsarrhythmia. The boy died of pneumo-
nia at the age of 13 years.
Bilateral PNH with frontonasal dysplasia
Seven patients (6 boys, 1 girl; age range: 5–22 years; mean
age: 11 years) had bilateral heterotopic nodules that were
diffuse, lining the lateral ventricles. Brain MRI showed multi-
ple cystic areas in the hemispheric white matter (Fig. 3C and
D). Partial agenesis of the corpus callosum was observed in
three. All seven patients had severe hypertelorism with inner
and outer canthal distances above the 97th percentile, broad
nasal root, poorly formed nasal tip, widow’s peak and mild
mental retardation; three had focal epilepsy. Two boys in this
group were reported in the original description of the
PNH frontonasal malformation syndrome (Guerrini and
Dobyns, 1998). The five additional patients had overlapping
characteristics, confirming the specificity of this syndrome.
All patients were sporadic. Mutation analysis of FLNA,
performed in six patients, gave negative results.
Bilateral PNH with limb abnormalities
Six patients (3 males, 3 females) had diffuse bilateral
PNH sparing the temporal horns, similar to classical bilateral
PNH (Fig.4A), associated
However, abnormalities of limbs had different characteris-
tics, possibly corresponding to two phenotypic subgroups.
Four patients (1 male, 3 female) had limb reduction abnor-
malities, with missing or hypoplastic phalanges of toes
Fig. 3 Brain imaging in four children with different forms
of PNH. In A, axial section showing bilateral PNH with
fronto-perisylvian polymicrogyria in a 4-year-old boy. Small
subependymal nodules are visible in both frontal and occipital
horns (arrows). There is polymicrogyria in the perisylvian and
fronto-opercular cortex bilaterally. In B, axial section in a
6-year-old boy with PNH and hydrocephalus. There is severe
enlargement of both lateral ventricles, especially involving the
occipital horns where small clusters of subependymal nodules are
present (arrowheads). C and D are coronal and axial sections in
two 7-years-old boys with bilateral PNH with frontonasal dysplasia.
Both children present bilateral contiguous subependymal
nodules (arrowheads) and structural abnormality in the white
matter where scattered cystic formations are present, possibly
representing dilated Virchow-Robin spaces.
Anatomoclinical spectrum of PH and FLNA mutations Brain (2006) Page 7 of 15
or fingers and of metatarsal or metacarpal bones (Fig. 4B).
These four patients had mild mental retardation and one had
epilepsy. Mutation analysis of FLNA, performed in two, was
negative. Twoboys had the
retardation–syndactyly syndrome (patients BPNH-03 and
BPNH-12 in Dobyns et al., 1997). Mutation analysis of
FLNA, performed in both, was negative. In previous studies,
a large duplication of Xq28 also containing FLNA, had been
demonstrated in a third boy with an identical phenotype, not
included in this series (patient BPNH-02 in Dobyns et al.,
1997 also reported by Fink et al., 1997 and Fox et al., 1998).
The same duplication was not identified in the two patients
reported here (Fink et al., 1997), prompting us to perform
mutation analysis. Further studies at the molecular karyo-
typing and intragenic level are in progress in these two
patients in order to identify deletions or duplications of
bilateral PNH, mental
Bilateral PNH with Ehlers–Danlos
Three unrelated women had classical bilateral PNH asso-
ciated with EDS (Fig. 1C and D). Two of them had epilepsy;
one had borderline cognitive level and the remaining two
had normal intelligence. Two of them were described in a
previous report (Sheen et al., 2005; patients F7 and F8),
while the third patient (5-II.1) is reported in detail
below (see Family 5). Mutation analysis of FLNA revealed
a single base deletion in two probands and a missense
mutation in one.
Bilateral PNH with fragile-X syndrome
Two boys with the fragile-X syndrome (age 13 years and
4 years) were found to have PNH on brain imaging. One
of them had small scattered bilateral subependymal nodules
as well as malrotated hippocampi; the second had a single
large nodule beneath the right lateral ventricle. Neither of
them had epilepsy. Southern blotting demonstrated a CGG
trinucleotide repeat expansion in the 50end of the Fragile site
mental retardation 1 (FMR1) gene in both boys.
Other syndromes with bilateral PNH
Two additional syndromes with bilateral PNH were less
clearly characterized and seen in a few patients. Two boys
had bilateral periventricular micronodular heterotopia, with
scattered subependymal nodules, each less than a few
millimetres thick. These nodules barely altered the ventri-
cular profile and could only be seen on high resolution MRI,
which was prompted by childhood onset seizures and mental
retardation in both patients. One additional individual with
ambiguous genitalia was born with pseudohermaphroditism,
hypospadia with adjacent uterine tubes and vagina. An
orchiectomy was performed and female hormonal therapy
given, and the child was raised as a girl. Her cognitive level
was normal. Standard karyotype was 46,XY and FISH
(fluorescent in situ hybridization) for subtelomeric imbal-
ances was negative. After onset of generalized seizures at
25 years, brain MRI revealed bilateral PNH. Mutation
analysis of FLNA was negative in all three patients.
Fig. 4 A and B illustrate PNH and severe limb limb reduction abnormality in a 2-year-old girl. In A, a coronal section shows non-contiguous
heterotopic nodules (arrowheads) and dilated ventricles. In B, the left hand is shown. In C, an axial section in a 15-year-old girl with diffuse
linear PH surrounding the lateral ventricles (arrowheads); no nodules can be seen. There is thickening of the cortex with simplified gyral
pattern especially in the frontal lobes. D and E show PH with ribbon-like aspect in a 25 years old woman. D is an axial section showing grey
matter heterotopia surrounding the posterior aspect of the lateral ventricles and E is a magnification showing the nearly sinusoidal ribbon
like structure of the heterotopic grey matter that is reminiscent of a simplified gyral pattern.
Page 8 of 15Brain (2006)E. Parrini et al.
Unilateral diffuse PNH sparing the
Fifteen patients (7 male; 8 female; age 3–36 years, mean age:
17 years) had unilateral PNH whose characteristics were
similar to those of classical bilateral PNH, although latera-
lized (Fig. 1B). No associated brain or extracerebral malfor-
mations were seen. Thirteen patients were sporadic with no
reported consanguinity; mutation analysis was negative in
8 of these patients. In one family, a father and daughter
had unilateral PNH on opposite sides (Guerrini et al.,
2004; patients 2-II.3 and 2-III.1). Mutation analysis of
FLNA demonstrated an S149P mutation in both individuals,
as well as in the asymptomatic proband’s paternal grand-
mother who refused brain MRI scanning. The S149P change
must therefore be germline at least in the two individuals
who inherited it (i.e. somatic mosaicism cannot account for
the unilateral presentation). Among the entire group, eight
patients had epilepsy; one had had infantile spasms and
all the remaining had focal epilepsy. Age at seizure onset
ranged from 1 to 38 years (mean 16 years). Seven patients
had normal cognitive level and eight had mild mental
Diffuse linear PH
Three unrelated children, two boys and a girl, had PH
characterized by a smooth layer of subependymal grey matter
rather than discrete or confluent nodules (Fig. 4C). The gyral
pattern was mildly simplified with areas of infolding and
abnormally thick cortex suggestive of a widespread malfor-
mation of neuronal migration. All three children had severe
developmental delay, mental retardation and epilepsy. Muta-
tion analysis of FLNA, performed in all, gave negative results.
PH with ribbon-like aspect
Two unrelated patients, a man and a woman in their early
adulthood, had ribbon-like PH encircling the posterior
bodies and occipital horns of the lateral ventricles.
On close inspection, the heterotopic ribbon appeared
convoluted with a nearly sinusoidal regularity reminiscent
of a simplified gyral pattern (Fig. 4D and E). There were no
associated malformations. Both patients presented with
childhood onset seizures that prompted brain imaging
studies, and had normal intelligence; both were sporadic.
Mutation analysis of FLNA was not performed. Mutation
analysis of the doublecortin (DCX) gene in one patient
gave negative results.
We found 25 mutations of the FLNA gene in 40 patients,
including 16 novel mutations. The latter consisted of
4 splice site mutations, 5 nonsense mutations, 5 deletions
and 2 insertions (Table 2, Fig. 6).
Twenty-five patients belonged to 10 families with X-linked
PNH and mutations were found in all of them (100%). Four
families with classical bilateral PNH (Family 1 and 2 in Moro
et al., 2002; Family 1 and 4 in Guerrini et al., 2004) and one
family with unilateral PNH (Family 2 in Guerrini et al.,
2004) were described in previous reports. Five unreported
families, including four with classical bilateral PNH and one
with EDS in the proband, are described in the following
section. Fifteen patients were sporadic: 13 had classical
bilateral PNH (including Patient 1-II.2 of Parrini et al.,
2004) and 2 had associated EDS (including Patients F7
and F8 of Sheen et al., 2005).
Overall, the rate of FLNA mutations was 49% in patients
with classical bilateral PNH, of which 33 were in 43 females
(77%) and 2 in 29 males (7%), irrespective of their being
familial or sporadic, but ranged from 100% in familial cases
(3 males, 19 females) to 26% in sporadic patients (13 out of
50 patients). In particular, the probability of finding FLNA
mutations in a sporadic individual with classical bilateral
PNH was 54% in females (13 out of 24) and 0% in males
(0 out of 26); while in individuals with other phenotypes it
was 8% (2 out of 24) in females and 0% (0 out of 24) in
males, limited to the 2 minor variants of EDS and unilateral
PNH. Thirty-seven out of 40 patients (93%) with mutations
of FLNA were female.
Family 1 (Table 2; individuals 2a and b): The proband
(Fig. 5, 1-II.3) was a 20-year-old woman with borderline
intelligence quotient (IQ) [Full Scale Intelligence Quotient
(FSIQ) = 69] and headache, who had an MRI scan following
episodes of delirium that were interpreted as of psychotic
origin. Her brain MRI and that of her 24-year-old sister
(Fig. 5, 1-II.4) revealed classical bilateral PNH. DHPLC
analysis of FLNA showed an abnormal elution profile for
exon 31, and sequencing revealed a c.5327 C!T nucleotide
substitution. The latter causes a G1710G silent mutation
that was predicted to be recognized as an alternative splice
acceptor site (BDGP splice database score = 0.99). To verify
the hypothesis of alternative splicing, a fragment spanning
700 bp of FLNA cDNA encompassing the mutation site
(from exon 31 to 32) was amplified from cDNA obtained
by RT–PCR from patient-derived lymphoblastoid cells, and
subsequently sequenced. The resulting cDNA sequence
(exons 31–32) was normal and did not show the nucleotide
variation identified in genomic DNA, suggesting that the
allele carrying the mutation was not correctly expressed.
Subcloned RT–PCR products showed that 1 out of 39 clones
contained a shorter aberrant mRNA spliced insert (352 bp
instead of 412 bp) with a new donor splice site created
by the c.5327 C!T nucleotide substitution. Both NMD
and skewed X-chromosome inactivation might explain the
underexpression of the mutated allele. NMD is an RNA
surveillance mechanism essential for maintaining mRNA
quality control by degrading mRNAs containing premature
termination codons (Holbrook et al., 2004). The RT–PCR,
Anatomoclinical spectrum of PH and FLNA mutationsBrain (2006)Page 9 of 15
subcloning and sequence analysis performed on the patient’s
lymphoblastoid cell line treated with cycloheximide showed
an increased number of clones (8 out of 35) containing the
aberrant mRNA splicing product, consistent with NMD
(Fig. A in the Supplementary online material). In addition,
X-inactivation studies in the patient’s lymphoblastoid cell
line revealed a significant skewing with an 80:20 ratio
(data not shown). These results indicate that both NMD
and skewed X-chromosome inactivation have contributed
to the prevalent expression of the normal allele in the
patient’s lymphoblastoid cell line.
Family 2 (Table 2; individuals 6a and b): The proband
(Fig. 5; 2-III.2) was a 15-year-old girl with idiopathic
thrombocytopenia, PDA, severe scoliosis, joint laxity and
dental malposition. Brain MRI (Fig. 1A) revealed classical
bilateral PNH in the proband and in her mother (Fig. 5;
2-II.2). DHPLC analysis showed an abnormal profile of exon
13 of the FLNA gene in the proband; sequencing analysis
revealed a c.2002 C!T nucleotide substitution in both
patients, leading to a protein truncation (Q668X).
Family 3 (Table 2; individuals 7a–d): The proband (Fig. 5;
3-II.3) was a 33-year-old woman with classical bilateral PNH
and epilepsy who had had four miscarriages. Both patients’
sisters (Fig. 5; 3-II.2 and 3-II.6) had classical bilateral PNH
and epilepsy; their mother (Fig. 5; 3-I.2) had classical bilat-
eral PNH but no epilepsy. DHPLC analysis in the proband
showed an abnormal profile of exon 25. Sequencing revealed
a substitution of 2 nt (c.[4437 A!G;4438 C!A]) in all
affected individuals, thus suggesting that the change was
in a cis configuration, leading to a TGA stop codon
(Y1479X). We confirmed the cis configuration subcloning
the PCR product of exon 25 in Patient 6a (Fig. B in the
Supplementary online material).
Family 4 (Table 2; individual 9a): The proband (Fig. 5;
4-II.1) was a 49-year-old woman with classical bilateral PNH
and epilepsy. Her 5-year-old daughter was also affected and
her mother was also probably affected as she had epilepsy but
refused MRI scan of the brain and DNA analysis. All three
individuals had normal intelligence. DHPLC analysis in the
proband showed an abnormal chromatographic profile of
exon 41. Sequencing detected a c.6724 C!T change, result-
ing in a stop codon and truncation of the protein at position
Eleven novel FLNA mutations were found in many sporadic
women with classical bilateral PNH (Table 2 and Fig. 6). We
found splice site mutations in three patients, truncating
mutations in two, deletions in four and insertions in two.
FLNA mutations in Ehlers–Danlos syndrome
The mutations associated with bilateral PNH and EDS in two
sporadic patients included in this series were previously
Fig. 5 Pedigrees of families 1–5.
Page 10 of 15Brain (2006) E. Parrini et al.
reported (c.2762 delG in exon 19 and A39G in exon 2; Sheen
et al., 2005), but we have new data on an additional patient.
Family 5 (Table 2; individuals 11a and b): The proband
(Fig. 5; 5-II.1) had bilateral PNH associated with EDS and
epilepsy, with joint hypermobility, soft hyperelastic skin with
widened paper-thin scars, dysmorphic facial features with
hypertelorism, hypoplastic midface, short nose, long shallow
philtrum, cupid’s bow upper lip and micrognathia. Her
intelligence was normal. Her mother (Fig. 5; 5-I.2) also
had epilepsy and bilateral PNH (Fig. 1C), but no dysmorphic
features or signs of EDS. DHPLC showed an abnormal pro-
file of exon 24 in the proband and sequencing revealed a
c.4038 delG in both patients, resulting in a frameshift and
presumed protein truncation.
Combining our results with previously reported mutations
of FLNA in patients with PNH, we found 14 mutations in the
ABD (904 nt; 10.2% of the gene), 17 in the Rod 1 + Hinge 1
domain (5029 nt; 56.7% of the gene), 8 in the Rod 2 + Hinge
2 domain (2629 nt; 29.7% of the gene), and 3 in the C-
terminal domain (299 nt; 3.4% of the gene). Statistical ana-
lysis using Fisher’s exact test indicated that the number of
FLNA mutations was significantly different in the ABD (P =
0.0087) compared to the number occurring in the three
remaining domains. Therefore, the ABD is a hotspot for
mutations causing bilateral PNH. No significant association
was found for the Rod 1 + Hinge 1, Rod 2 + Hinge 2 or C-
terminal domains (P > 0.05 for all three).
The x2test indicated that the sex ratio in the 37 patients
with classical bilateral PNH without FLNA mutations was
significantly skewed towards males (26 males and 11 females:
P = 0.013).
The main aim of our analysis was to offer new insights into
the nosology of PH, for optimizing genetic counselling and
future research. Our study examined the clinical and brain
MRI characteristics of 182 individuals with PH, including
bilateral PNH and other types of PH and led to the definition
of 15 distinct malformation patterns (Table 1). The overall
information that can be drawn from this study is that PH is
an extremely heterogeneous disorder regarding both clinical
and brain imaging presentation and genetic causes. Classical
bilateral PNH represented the largest group (98 patients:
54%). In most of the 15 additional phenotypes (84 patients:
46%), PH was associated with other brain malformations,
including hippocampal malformation and cerebellar hypo-
plasia, bilateralfronto-perisylvian or temporo-parieto-
occipital PMG, hydrocephalus and microcephaly. However,
it was possible to identify a smaller group of patients
in whomPH was associated
defects including EDS, frontonasal dysplasia, limb abnorm-
Finally, severaldistinct subgroups
identified in whom the PH presented an unusual appearance,
Fig. 6 Location of FLNA mutations (see Table 2 and Table A in Supplementary online material) on the structure of the FLNA monomer
containing repeat blocks of 96 amino acids (Gorlin et al., 1990). In the upper part of the panel are shown FLNA missense mutations associated
with periventricular heterotopia (PH). In the bottom part of the panel are shown FLNA mutations causing frame-shift or protein truncation
(nonsense, splice-site, deletions and insertions) that are associated with PH.§Known FLNA mutations also observed in this study; *new FLNA
mutations reported in this study; familial cases in this study;#EDS associated mutations.
Anatomoclinical spectrum of PH and FLNA mutationsBrain (2006) Page 11 of 15
including: micronodular appearance, unilateral distribution
and laminar or ribbon-like shapes.
Phenotypes associated with FLNA
FLNA appears to be the major gene associated with PH. In
this study, FLNA mutations were associated with the most
common phenotype, classical bilateral PNH, and with two
minor variants: unilateral PNH and bilateral PNH with EDS.
FLNA maps to Xq28 and codes for the high molecular mass
protein filamin A which mediates crucial processes for spatial
and temporal coordination of cell reshaping and motility.
Although X-linked and sporadic PNH has been associated
with FLNA mutations (Fox et al., 1998), PH is expected to be
genetically heterogeneous. For instance, recessive PH with
microcephaly has been associated with ARFGEF2 mutations
(Sheen et al., 2003a; Sheen et al., 2004a).
Mutation analysis of FLNA in a cohort of 120 probands
from our total group of 182 with PH (?50% males and
?50% females) uncovered 25 FLNA mutations of which
16 are novel. Consistent with prior reports, the sex ratio
among these patients is skewed toward females: 93% of
patients harbouring FLNA mutations were female and 7%
were males. Mutations of FLNA were found in all familial
cases of classical X-linked bilateral PNH (8 families;
22 affected individuals) as previously reported (Sheen et al.,
2004a), while about 26% of sporadic patients, all females,
with classical bilateral PNH harbour an FLNA mutation.
Overall, the probability of identifying a mutation in an
individual with classical bilateral PNH was 49%, but this
decreased to 4% in patients with other phenotypes, irrespec-
tive of their being familial or sporadic. We identified addi-
tional FLNA mutations only in patients with unilateral PH
and with bilateral PNH associated with EDS. Thus, the cause
of the PH remained unknown in 51% of patients with clas-
sical bilateral PNH, and in 96% of those with other
PH phenotypes, confirming causal heterogeneity of PH.
We believe that all or most of the remaining causes are
genetic based on reports of other loci (dup 5p15.1 and
dup 5p15.33) and genes (ARFGEF2), skewing of the sex
ratio in at least two syndromes, and lack of evidence sup-
porting extrinsic causes (Sheen et al., 2003b; Sheen et al.,
2004a; Wieck et al., 2005).
A family with unilateral PH and a missense mutation
(S149P) had already been reported (Family 2 in Guerrini
et al., 2004). In this family, we hypothesized the mild
male phenotype and father to daughter transmission to be
consistent with mild functional impairment of the FLNA
A previous description of PNH with EDS (Sheen et al.,
2005) included the missense mutation and truncating
mutation in the two patients also described here (Patients
F7 and F8 in Sheen et al., 2005). In the present study, we have
also identified an FLNA deletion (c.4038 delG) in a family
(Family 5) in which the proband (5-II.2) had PNH with EDS
but her mother (5-I.2) had only classical bilateral PNH,
suggesting that the clinical features of EDS may reflect
variable expressivity, most likely due to genetic modifying
factors. Phenotypic heterogeneity might also result from
skewed X-inactivation in either woman or somatic mosai-
cism in the mother. The association between PNH and EDS
changes the understanding of the molecular pathogenesis of
EDS. Generally, different types of EDS have been associated
with alterations of the cross-linkage and adhesion of collagen
fibrils in the extracellular matrix (Sheen et al., 2005). FLNA
has been shown to bind beta integrin cell adhesion receptors
and this interaction is possibly involved in cell migration
(Sharma et al., 1995; Calderwood et al., 2001). Impaired
cellular adhesion due to FLNA mutations might therefore
be responsible for both the defects in connective tissue seen
in EDS and failure in neuronal migration from the ventri-
cular zone, which is typical of PNH. Overall, only 3 out of
the 40 patients (7.5 %) reported here with PNH and an
FLNA mutation also had EDS. Mutations identified in
these patients did not cluster in any specific FLNA region
and were predicted to cause variable functional consequences
in the protein product. In addition, in most patients with the
PNH-EDS phenotype no FLNA mutations can be demon-
strated (Sheen et al., 2005). These observations leave the
genetic basis of the PNH-EDS phenotype unexplained.
This study brings to 42 the number of mutations of
FLNA so far described in association with PNH, with or
without EDS (Fig. 6). Considering all 42 FLNA mutations
identified (Fig. 6), the prevalence of mutations in the ABD
was significantly elevated (14 out of 42 in 904 nt) (P < 0.05)
compared with the prevalence of mutations in the other
three domains (28 out of 42 in 7957 nt) (P > 0.05). This
result suggests that the ABD is a hotspot for FLNA mutations
causing PNH. Therefore, mutation analysis of exons encom-
passing the ABD should be performed first in these patients.
Some regions of FLNA have been identified that are
associated with other specific disorders. Missense mutations
falling within the CH2 domain and rod-domain repeats 3,
10 or 14/15 were observed in males with OPD1 or OPD2
(Robertson et al., 2003). However, no mutations leading to
OPD1 or OPD2 fell within the CH1 domain. All four mis-
sense mutations we found fell within the CH1 domain and
none in the CH2 domain. Overall, no missense mutations in
CH2 have been identified in patients with PNH (Fig. 6) but
several early truncating mutations causing loss of the CH2
domain have been detected in patients with PNH without
the OPD phenotype. This observation confirms previous
suggestions that missense mutations in CH1 in patients
with PNH cause loss of function, while missense mutations
in CH2, which are always associated with the OPD spectrum,
cause a gain of function (Sheen et al., 2001; Robertson
et al., 2003).
Overall, only 9 of the 42 FLNA mutations that have so far
been associated with PNH are missense mutations, suggest-
ing that mutations causing protein truncation are the main
cause of the PNH phenotype. This contrasts sharply with the
Page 12 of 15 Brain (2006)E. Parrini et al.
OPD syndromes that have been associated only with mis-
sense mutations (Robertson et al., 2003). This observation
confirms that distinct pathogenic mechanisms underlie
these two different phenotypes.
In general, some correlation between the severity of
FLNA mutations and the associated PNH phenotype
seems to exist but is not yet clear even with this large number
of mutations. In our study we did not observe significant
differences between the type and location of mutations, and
the severity of the associated phenotype. One exception is
represented by missense germline mutations and distal trun-
cating mutations that are compatible with survival of affected
males, while insertions, deletions or truncating mutations
are lethal in males. Partial or residual function of the protein
presumably accounts for male viability (Sheen et al., 2001;
Guerrini et al., 2004). Somatic mosaicism in patients with
truncating mutations may also attenuate the phenotype
(Guerrini et al., 2004; Parrini et al., 2004).
Classical bilateral PNH not associated
with FLNA mutations
Classical bilateral PNH was observed in 37 (51%) of the
patients in whom FLNA mutations were not found. These
patients did not show significant clinical and imaging differ-
ences with respect to those with mutations. Mutations in
non-coding regions, larger deletions/duplications involving
one or more exons, and cryptic chromosomal abnormalities
involving FLNA may still account for some of the patients in
whom no mutations were detected. However, sex ratio in
this group reached statistical significance towards male
predominance (P = 0.013), suggesting that an alternative
X-linked recessive gene may play a major role. However,
some ascertainment bias due to a higher proportion of
males being referred due to their more severe phenotype
cannot be excluded.
Major phenotypes not associated
with FLNA mutations
None of the patients belonging to these groups harboured
mutations in FLNA, suggesting that other genes are likely
to play a major role. However, FLNA cannot be completely
ruled out as the disease gene as mutations in non-coding
regions, larger deletions/duplications involving one or more
exons, and cryptic chromosomal abnormalities involving
FLNA might still be responsible for these phenotypes.
Amongst patients with bilateral PNH associated with non-
neurological defects, at least four groups were homogeneous
and large enough to be considered as new syndromes or to
confirm previously hypothesized syndromes. Patients with
bilateral frontal-perisylvian PNH-PMG, bilateral posterior
PNH-PMG, and those with autosomal recessive bilateral
PNH and microcephaly have been the subject of separate
reports and will not be discussed further here (Sheen et al.,
2004a; Wieck et al., 2005). Another syndrome consisting of
severe congenital microcephaly, diffuse PNH and diffuse
PMG was reported in two patients (Wieck et al., 2005),
but not seen in this series.
Bilateral posterior PNH with hippocampal malformation
and cerebellar hypoplasia represents a newly recognized
syndrome found in more females than males, but without
significant skewing of the sex ratio in this first small group of
ten patients. It must be differentiated from classical bilateral
PNH due to FLNA mutations. The main difference is in the
location of the heterotopic nodules, which in classical bilat-
eral PNH are diffuse but do not extend into the temporal
horns and do not usually surround the hippocampal forma-
tion. Incomplete hippocampal rotation is often observed in
patients with neuronal migration disorders (Baulac et al.,
1998) but may also occur as an isolated abnormality
(Fernandez et al., 1998). In the patients described here,
malrotated hipocampi are associated with abnormal neuro-
nal migration in the parahippocampal cortex and in mesial
temporal structures. Patients in this group had severely
hypoplastic cerebellum. Vermis hypoplasia with mega
cisterna magna is also present in some patients with classical
bilateral PNH due to FLNA mutations (Fox et al., 1998) but
is never as severe as in the patients reported here, for most of
whom cerebellar signs prompted neuroradiological investi-
gations. Cognitive level and types of epilepsy did not differ
from what is usually observed in classical bilateral PNH.
Bilateral posterior PNH with hydrocephalus was observed
in seven patients, most having severe developmental delay
and epilepsy. The sex ratio in this group was balanced
suggesting an autosomal pattern of inheritance. Sheen
et al. (2004b) reported weakly positive linkage to Xq28 in
one family, but no mutations were found in FLNA or in
L1CAM (L1 cell adhesion molecule), a gene associated with
hydrocephalus, Hirschsprung disease and agenesis of the
corpus callosum. While the PNH-hydrocephalus phenotype
is quite homogeneous, its genetic basis is probably
Bilateral PNH with frontonasal dysplasia was observed in
six sporadic boys and one girl. Mild mental retardation was
present in all and epilepsy in three. Two of the boys had been
previously described (Guerrini and Dobyns, 1998). The five
additional patients included in the present study confirm the
specificity of the syndrome and bring to six males and two
females (seven cases included herein and one girl reported by
Guion-Almeida and Richieri-Costa, 1999) the total number
of patients described so far. Skewing of gender ratio does not
reach statistical significance (Fisher’s exact test: P > 0.05).
Bilateral PNH with limb abnormalities was observed in six
patients; all had mental retardation and two had epilepsy.
Severity of limb abnormalities was variable, ranging from
severe limb reduction with missing or hypoplastic phalanges
of fingers and toes to syndactyly. All patients were sporadic
and gender ratio in this group was balanced. Recessive
or ‘de novo’ dominant mutations are both possible. Genes
regulating limb development are possible candidates for this
syndrome. In particular FGF8, involved in the FGF (fibro-
blast growth factor) signalling from the apical ectodermal
Anatomoclinical spectrum of PH and FLNA mutations Brain (2006)Page 13 of 15
ridge (Lewandoski et al., 2000), could be a good candidate as
there is evidence that it is involved in neocortical patterning
(Fukuchi-Shimogori and Grove, 2003).
Minor phenotypes with PH
We identified five additional rare conditions with PH. No
mutations of FLNA were identified in these small subgroups
of patients but not all of them were tested. These observa-
tions confirm the phenotypic and genotypic diversity asso-
ciated with PH.
Fragile-X syndrome had been diagnosed in two boys with
isolated or scattered PH and a full mutation of FMR1, sug-
gesting a possible role of this gene in neuronal migration.
Bilateral periventricular micronodular heterotopia and bilat-
eral PNH with ambiguous genitalia were observed in three
patients only and are less clearly characterized.
Diffuse linear PH occurred in association with more
widespread cortical thickening suggesting a generalized
migration abnormality differently affecting migrating and
non-migrating neurons. PH with ribbon-like shape occurred
as an isolated abnormality affecting a subset of abnormally
migrating neurons that although receiving the genetic infor-
mation to assemble in a convoluted, cortical-like pattern,
were not able to reach their final destination.
The Supplementary data are available at Brain on-line.
We thank the patients, their families, and the referring
physicians. C.A.W. is an Investigator of the Howard Hughes
Medical Institute. This study complies with the current
Italian laws and was supported with funding from Fonda-
zione Pierfranco e Luisa Mariani (grant R-04-35), the Italian
Ministry of Health (grant RF 2/02) and grants from the
NINDS (R37 NS35129) to C.A.W.
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