HUMAN MUTATION 28(4),356^364,2007
Truncation of NHEJ1 in a Patient
Vincent Cantagrel,1,2Anne-Marie Lossi,1,2Steven Lisgo,3Chantal Missirian,1,4Ana Borges,1,2
Nicole Philip,1,4Carla Fernandez,5Carlos Cardoso,1,2Dominique Figarella-Branger,5
Anne Moncla,1,4Susan Lindsay,3William B. Dobyns,6–8and Laurent Villard1,2?
1INSERM, U491, Faculte ´ de Me ´decine La Timone, Marseille, France;2Aix Marseille Universite ´, Faculte ´ de Me ´decine, Marseille, France;
3Institute of Human Genetics, International Centre for Life, Newcastle, United Kingdom;4Department of Medical Genetics, La Timone Children’s
Hospital, Marseille, France;5Nervous et Muscular Biopathology, Faculte ´ de Me ´decine La Timone, Marseille, France;6Department of Human
Genetics, The University of Chicago, Chicago, Illinois;7Department of Neurology, The University of Chicago, Chicago, Illinois;8Department of
Pediatrics, The University of Chicago, Chicago, Illinois
Communicated by Maria Rita Passos-Bueno
Polymicrogyria (PMG) is a common malformation of the human cerebral cortex for which both acquired and
genetic causes are known. Although genetic heterogeneity is documented, only one gene is currently known to
cause isolated PMG. To clone new genes involved in this type of cerebral malformation, we studied a fetus
presenting a defect of cortical organization consisting of a polymicrogyric cortex and neuronal heterotopia
within the white matter. Karyotype analysis revealed that the fetus was carrier of a balanced, de novo,
chromosomal translocation t(2;7)(q35;p22). Cloning and sequencing of the two translocation breakpoints
reveals that the chromosomal rearrangement disrupts the coding region of a single gene, called NHEJ1,
Cernunnos, or XLF, in 2q35. The NHEJ1 gene was recently identified as being responsible for autosomal
recessive immunodeficiency with microcephaly. Using quantitative PCR experiments, we show that a truncated
transcript is expressed in the polymicrogyric patient cells, suggesting a potential dominant negative effect
possibly leading to a different phenotype. We performed in situ hybridization on human embryos and showed
that the NHEJ1 transcript is preferentially expressed in the telencephalic ventricular and subventricular zones,
consistent with the phenotype of the affected individual. In the human adult central nervous system (CNS),
NHEJ1 is mainly expressed in the cerebral cortex and in the cerebellum. The association of PMG with the
disruption of its transcript suggests that, in addition to its recently uncovered function in the immune system,
the NHEJ1 protein may also play a role during development of the human cerebral cortex. Hum Mutat 28(4),
rrrr2006 Wiley-Liss, Inc.
KEY WORDS: polymicrogyria; human embryo; XLF; Cernunnos; NHEJ1; cortical dysplasia
Malformations of cortical development, also called cortical
dysplasia, are a common cause of epilepsy and cognitive
deficits. Recent estimates have suggested that up to 25% of
adults and 40% of children with intractable epilepsy have
cortical dysplasia [Kuzniecky and Jackson, 1995; Meencke and
Veith, 1992; Vinters et al., 1992]. Polymicrogyria (PMG) is a
well-known and relatively common malformation of cortical
loss of the normal gyral pattern, which is replaced by many
small and infolded gyri separated by shallow sulci that are
often partly fused in their depths, and reduction of the normal
six-layered cortex to a four-layered or unlayered cortex. Affected
individuals present with variable phenotypes that range from
severe neonatal encephalopathies to normal intelligence with
specific cognitive defects, depending on the extent and specific
location of PMG, as well as overall head size, although most
patients have mental retardation [Guerrini et al., 2003]. Seizures
typically begin between 4 and 12 years of age and are often
Despite many descriptions, the pathogenesis of PMG remains
poorly understood, and is likely to be heterogeneous. Older reports
support extrinsic causes, especially vascular causes such as
intrauterine cytomegalovirus infection and placental perfusion
problems as may occur with twinning, and point to a period of risk
between 13 and 24 weeks gestation [Barkovich and Lindan, 1994;
Barth, 1992; Iannetti et al., 1998]. Substantial other evidence
supports a genetic basis for PMG including reports of familial
recurrence consistent with autosomal or X-linked inheritance
Published online 26 December 2006 in Wiley InterScience (www.
?Correspondence to: Laurent Villard, INSERM U491, FaculteŁ de
MeŁdecine de LaTimone,27 Bd. Jean Moulin,13385 Marseille Cedex
5, France. E-mail: email@example.com
July 2006;acceptedrevised manuscript 17
Grant sponsor: INSERM; Grant sponsor: Programme Hospitalier
de Recherche Clinique (PHRC National 2004); Grant sponsor: le
Groupement d’InteŁreŒt Scienti¢que (GIS) Institut desMaladies Rares.
rrrr2006 WILEY-LISS, INC.
[reviewed in Jansen and Andermann, 2005], our report of a PMG
locus in Xq28 [Villard et al., 2002], and association with several
chromosome abnormalities, particularly involving the DiGeorge
syndrome critical region in chromosome 22q11.2 [Robin et al.,
2006]. PMG has also been reported in several multiple congenital
anomaly syndromes, such as Adams-Oliver (MIM] 100300),
Aicardi (MIM] 304050), Goldberg-Shprintzen (MIM] 182212),
(Warburg) Micro (MIM] 600118), and oculocerebrocutaneous
(Delleman) (MIM] 164180) syndromes. However, mutations of
only a few genes have been associated with PMG. The first is
RAB3GAP , which regulates the Rab3 pathway implicated in
exocytic release of neurotransmitters, hormones, and possibly
trophic factors, and is mutated in some patients with Micro
syndrome [Aligianis et al., 2005]. The next is KIAA1279, a gene of
unknown function that is mutated in Goldberg-Shprintzen syndrome
[Brooks et al., 2005]. Finally, both heterozygous and especially
homozygous mutations of PAX6 have been associated with PMG
[Glaser et al., 1994; Mitchell et al., 2003].
In addition, several atypical cortical malformations that grossly
resemble PMG but differ on detailed review have been described in
neonatal adrenoleukodystrophy and Zellweger syndrome [Evrard
et al., 1978; Kelley et al., 1986], the cobblestone cortical
malformations found in Fukuyama congenital muscular dystrophy,
muscle-eye-brain and Walker-Warburg syndromes [Dobyns et al.,
1989; Haltia et al., 1997; Takada et al., 1988], and cobblestone-like
changes associated with mutations of the GPR56 and SNAP29 genes
[Chang et al., 2003; Piao et al., 2004, 2005; Sprecher et al., 2005].
In our experience, informative families useful for mapping
Mendelian forms of typical PMG are rare. An alternative strategy
to identify disease genes is to take advantage of de novo
chromosomal rearrangements in affected patients. This strategy
has led to the identification of a large number of genes, including
genes implicated in different types of cortical dysplasia such as
lissencephaly either due to LIS1 [Lo Nigro et al., 1997] or DCX
mutations [Gleeson et al., 1998], or periventricular nodular
heterotopia due to FLNA mutations [Fox et al., 1998].
We studied a fetus with a brain malformation consisting of diffuse
PMG and small mostly submicroscopic heterotopia distributed
throughout the white matter. Chromosome analysis demonstrated
an apparently balanced, chromosomal translocation: 46,XX,
t(2;7)(q35;p22) de novo. We showed that the rearrangement is
molecularly balanced and that it disrupts the coding region of the
in 2q35. NHEJ1 encodes a very recently described 33-kDa protein
that is a component of the mammalian DNA nonhomologous end-
joining (NHEJ) apparatus [Ahnesorg et al., 2006; Buck et al.,
2006]. The NHEJ1 protein interacts with XRCC4 and promotes the
repair of double strand breaks in mammalian cells [Callebaut et al.,
2006]. Mutations in this gene are found in patients affected by an
autosomal recessive form of immunodeficiency with microcephaly.
The expression of this gene during development has not been
previously described. We performed in situ hybridization on human
embryos and show that the NHEJ1 transcript is preferentially
expressed in the telencephalic ventricular and subventricular zones,
consistent with the phenotype of the affected individual. We also
show that a truncated transcript is expressed in the patient’s cells.
Because NHEJ1 needs to form homo- and heterodimers to function
properly, we discuss the possibility that the phenotype of our patient
is the result of a dominant negative effect. The association of PMG
with the disruption of this transcript suggests that, in addition to its
recently uncovered function in the immune system, the NHEJ1
gene may also play a role during development of the human cerebral
XLF, or FLJ12610) gene
Fluorescent In Situ Hybridization (FISH)
on Metaphase Chromosomes
BAC DNA prepared according to standard procedures was
labeled by nick translation. The reactions were carried out in a
volume of 50mL with 1mg of DNA, 50mM Tris Hcl, 5mM MgCl2,
10mM b-mercaptoethanol, 10mg/mL bovine serum albumin
(BSA), 50mM dATP , dCTP and dGTP , 30mM of dTTP , 20mM
of dUTP labeled with Spectrum Green or Spectrum Orange (Vysis,
Downers Grove, IL; www.vysis.com), and 2.5U of DNA
polymerase I/Dnase I mix (Invitrogen, Carlsbad, CA; www.
invitrogen.com). The reactions were incubated at 151C for 45
minutes and 16mg of human Cot1 DNA and 800mg of salmon
sperm DNA were added before ethanol precipitation. The dry
pellets were resuspended with the hybridization buffer (formamide
50%, 2? salt saline citrate (SSC), SDS 10%, sulfate dextran 10%,
Denhardt’s buffer 1?, pH 7). Hybridization was performed as
previously described [Chong et al., 1997]. Chromosomes were
counterstained with 40,60-diamino-2-phenylindole (DAPI) and
observed using a Zeiss Axiophot microscope. Images were
collected using a charge-coupled device (CCD) camera (Sensys-
Photometrics, Tucson, AZ; www.photomet.com/support_sensys.
html) and Quips Lab manager (Vysis).
Cell Culture, RNA Isolation, and Reverse Transcription
All lymphoblastoid cell lines were grown in RPMI 1680
(Invitrogen) with 10% fetal bovine serum in the presence of
0.1mg/ml of kanamycin at 371C and 5% CO2. Total RNA was
extracted from patient’s cells using TRIzol reagent (Invitrogen).
The RNA preparations were analyzed for purity using the
www.nanodrop.com). DNAse treatment was performed for 30
minutes with 5mg of RNA, 5U of RNAse-free DNAse I (Roche,
Basel, Switzerland; www.roche-diagnostics.com/researchers/mole-
cularbiology.html). Human normal tissue RNA was purchased (BD
Biosciences, San Jose, CA; www.bdbiosciences.com). Reverse
transcription of 5mg of total RNA was performed in 50ml of 1?
Superscript reaction buffer (Invitrogen) containing 3ng/ml of dN6,
40U of RNasin (Promega, Madison, WI; www.promega.com/),
10mM dNTP , and 200U of Superscript II reverse transcriptase
(Invitrogen). For every reaction, one RNA sample was treated
similarly without Superscript II RT (RT-reaction) to provide a
Northern and Southern Blot Hybridizations
We hybridized human fetal MTN blot II and human brain MTN
blot II (BD Biosciences) with a NHEJ1 cDNA probe (nucleotides
78–1061 of the cDNA sequence BC030986) and a probe for
b-actin (BD Bioscience,). These probes were labeled by random
priming using [a-32P]dCTP . Hybridization of Northern blots were
carried out in 50% formamide buffer at 421C for 16hr. For
Southern blot preparation, DNA samples were digested with HpaI,
electrophoresed on 1% agarose gel, and blotted onto Hybond N1
nylon membrane (GE Healthcare, Chalfont St-Giles, UK; www.
gehealthcare.com). Hybridization and washing were carried
out, respectively, in 5? SSC/0.5% SDS/ 1? Denhardt’s buffer,
SSC/0.1% SDS at 651C according to standard
Classical and Real-Time PCR
Classical PCR reactions were performed in the T1 thermocycler
(Whatman-Biometra, Goettingen, Germany; www.biometra.de) in
HUMAN MUTATION 28(4),356^364,2007 357
Human Mutation DOI 10.1002/humu
a total volume of 50ml, containing 1? PCR buffer, 0.2mM
dNTPs, 2mM MgCl2, and 1U Taq polymerase (Invitrogen).
Real-time PCR reactions were performed in the LightCycler 480
system (Roche) using the SYBR Green I Master Kit (Roche) with
1mL cDNA (1/5 dilution of the first strand reaction), 100 to
300nM of each primer. Each reaction was performed in duplicate.
The results were imported and analyzed using Excel (Microsoft
Corporation, Redmond, WA; www.microsoft.com).
A total of 85 patients with PMG were investigated: 10 patients
with PMG and periventricular nodular heterotopia, three patients
withPMG and subcortical
patients with bilateral perisylvian PMG. In each case, DNA from
affected individuals was used for direct sequencing of NHEJ1. We
designed primer pairs for each of the seven coding exons including
primers: exon 2 (459-bp product) forward (50-GGAGAACGTA
AATCCATGCC-30) and reverse (30-CCTGTACTTCTGCCTGG
AAT-50); exon 3 (389-bp product) forward (50-TCTCATGTCCA
TTGCCTTCG-30) and reverse (30-GAGAGGAAGCTTTAGGT
GCT-50); exon 4 (299-bp product) forward (50-CTGGTACC
TAATACTGAATTGG-30) and reverse (30-GGGAGACTCATT
TGCATTAGA-50); exon 5 (358-bp product) forward (50-ACTGA
ACTAGGTGTCTCTGC-30) and reverse (30-GGCTTGAGATG
TACTTAGGG-50); exon 6 (328-bp product) forward (50-CCAGG
CTTGGATATGGTCG-30) and reverse (30-GGATCTAGGTTTA
TGCTGTCC-50); exon 7 (294-bp product) forward (50-ATCAGA
ATGCTTGGTGAATGC-30) and reverse (30-GGAATTGCTGCT
We used thefollowing
TCATTGG-50); exon 8 (377-bp product) forward (50-CTCATCC
TAACCAGGAGCC-30) and reverse (30-GGAAGCCAGTCTCT
GAGAAT-50). We sequenced all exons in both forward and reverse
directions. Sequencing was carried out by GATC Biotech
and Sequencer software (Gene Codes, Ann Arbor, MI; www.
genecodes.com) was used to analyze sequences and chromatograms.
Electronic Database Information
GenBank accession numbers for BAC clones are as follows:
AC068946 (RP11-803J6) and AC073316 (RP11-191P7). Geno-
mic clones were obtained from the Sanger Centre (Cambridge-
shire, England, UK; now called the Wellcome Trust Sanger
Institute). BAC contigs were built in silico using the Institute
for Genomic Research (TIGR) BAC end-sequence database
and NIX analysis at the United Kingdom Human Genome
Mapping Project (UK HGMP; www.hgmp.mrc.ac.uk/Registered/
Webapp/nix). Sequence analysis of the BACs overlapping and near
the breakpoints was performed with database tools and gene
prediction software available in the NIX interface. The NCBI Map
viewer (www.ncbi.nlm.nih.gov/mapview) was also used to study
the genomic regions interrupted by the chromosomal breakpoints.
Human embryos were collected following British national
guidelines [Polkinghorne, 1989] from terminated pregnancy
material, with appropriate maternal written consent and approval
from the Newcastle and North Tyneside National Health Service
FIGURE 1. Photomicrograph from the fetal polymicrogyric brain. A,B: Photomicrograph of polymicrogyric cortex shows multiple
shallow sulci and fusion of the molecular layer inside gyri (asterisks). C: Photomicrograph of the white matter shows heterotopic
neurons (arrow). [Color ¢gurecanbeviewedin theonlineissue,whichis available at www.interscience.wiley.com.]
358HUMAN MUTATION 28(4),356^364,2007
Human Mutation DOI 10.1002/humu
(NHS) Health Authority Joint Ethics Committee. The embryos
were obtained from the Medical Research Council (MRC)–
Wellcome Trust Human Developmental Biology Resource (HDBR;
www.hdbr.org) at Newcastle. Embryos were collected into cold
phosphate buffered saline (PBS), separated from surrounding
tissue, and fixed overnight in 4% paraformaldehyde at 41C, before
short-term storage at 41C in 70% ethanol. Placental tissue for
karyotype analysis was sampled prior to fixation of the embryo
tissue. The stage of development was assessed on the basis of
external features according to the Carnegie staging protocol
[O’Rahilly and Muller, 1987] modified for use with individual
embryos rather than in comparisons of many embryos simulta-
neously [Bullen and Wilson, 1997; Bullen et al., 1998].
Tissue In Situ Hybridization
Digoxigenin-labeled sense and antisense probes to NHEJ1 were
generated by in vitro transcription of linearized plasmid DNA
and purified through a ProbeQuant G-50 micro column (GE
Healthcare). The probes were hybridized at 681C using DIG
Easy Hyb (Roche) following the method outlined in Franco et al.
 using an 8-minute proteinase K pretreatment to 8-mm
tissue sections. Signal was detected by nitro blue tetrazolium/
5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) following
posthybridization washes and incubation with an antidigoxygenin,
alkaline phosphatase conjugated antibody.
This female fetus came to medical attention at 27 weeks
gestation when sonography demonstrated fetal hydrocephalus.
A karyotype was performed on cultured amniocytes, and
revealed a de novo apparently balanced translocation: 46,XX,
t(2;7)(q36;p22). The pregnancy was terminated at 33 weeks based
on these observations. At autopsy, growth parameters were
normal for gestational age (weight 2,490kg, height 46cm,
External and visceral examination was normal except for the
presence of a partial syndactyly of the fingers and complete
syndactyly of toes two to four. Routine histology analysis showed
no evidence of infection or inflammation. Examination of the
brain confirmed the presence of hydrocephalus, and also
demonstrated extensive PMG that appeared most severe in the
occipital lobes and to a lesser extent the frontal lobes (Fig. 1A and B).
Histological examination demonstrated a four-layered cortex
comprised of a molecular layer, a layer of granular neurons,
a poorly defined layer with few cells, and finally a layer
of disorganized pyramidal neurons, as well as excess vasculariza-
tion. The white matter demonstrated small groups of heterotopic
neurons throughout with a prevalence in the white matter
located beneath the polymicrogyric areas (Fig. 1C) as well as
periventricular astrocytic gliosis. Cytomegalovirus serology was
negative in the mother of this fetus.
[OFC] 33cm, foot 65mm).
Cytogenetic and Molecular Characterization
We first constructed a genomic contig with BAC clones
covering the region likely to contain the breakpoints based on
the karyotype observations (Fig. 2A). These clones were used as
probes in FISH experiments on metaphase chromosome spreads
from a lymphoblastoid cell line derived from the fetus. These serial
hybridizations led to the identification of two clones spanning the
translocation breakpoints: BAC RP11-803J6 in 2q35 and BAC
RP11-1228A3 in 7p22 (Fig. 2B and C). The 2q35 breakpoint was
more precisely mapped using Southern blot hybridizations. Serial
hybridizations were performed with probes originating from the
RP11-803J6 BAC clone until an aberrant restriction fragment was
detected. Using one such probe a patient-specific restriction
fragment was observed (Fig. 2D). This junction fragment was
cloned and sequenced. Sequence analysis showed that it originates
from 2q35 on one side and 7p22 on the other side. This data
allowed us to amplify and sequence the translocation breakpoints
on both derivative chromosomes and to show that the transloca-
tion is almost perfectly balanced (Fig. 2E). Indeed, comparison
with wild-type sequences revealed an insertion of 11bp on the
FIGURE 2. Cytogenetic and molecular characterization of the
breakpoints. A: Pictograms showing normal chromosomes 2
and 7 and their derivatives der(2) and der(7). B,C: Metaphase
chromosomes of the fetus were used for FISH experiments with
di¡erent clones spanning each breakpoint on der(2) and der(7):
RP11-803J6 clone (signal in red) from chromosome 2 (B) and
RP11-1228A3 clone (signal in red) from chromosome 7 (C).
A centromeric BAC from chromosome 7 (signal in green) (B)
and a telomeric BAC from short-arm of chromosome 2 (signal
in green) (C) were used as control probes. D: Southern-blot
hybridization of proband (P) and control (C) genomic DNA
digested by HpaI with a probe corresponding to nucleotides
36989^37412 of clone RP11-803J6. E: PCR ampli¢cation
of the two breakpoint-containing DNA fragments using the pro-
band genomic DNA, a control DNA, or no DNA using primers
originating from the two derivative chromosomes. Sequence of
HUMAN MUTATION 28(4),356^364,2007359
Human Mutation DOI 10.1002/humu
derivative chromosome 2 and no insertion or deletion on the
derivative chromosome 7 (data not shown). The 2q35 breakpoint
is located at position 39387 in the sequence of clone RP11-803J6
and the 7p22 breakpoint at position 29861 in the sequence of
clone RP11-191P7 (Fig. 3).
Sequence analysis using the NIX interface at HGMP indicates
that the chromosome 7 breakpoint does not interrupt a known
gene or anonymous expressed sequence tag (EST). The closest
known genes are the CARD11 gene located 140kb telomeric to
the breakpoint and the SDK1 gene located 107kb centromeric to
the breakpoint. A cluster of spliced ESTs (UniGene Hs.527001)
is detected 35kb telomeric to the breakpoint. We failed to detect
a corresponding transcript using RT-PCR in different tissues
including fetal brain (data not shown). SDK1 is expressed in fetal
brain and in several other tissues but it is not expressed
in lymphoblastoid cell lines (Fig. 4A). CARD11 is only weakly
expressed in brain but is expressed in lymphoblastoid cell lines.
We used a known SNP in the CARD11 transcript for which the
fetus was heterozygous and showed that the two CARD11 alleles
are expressed in the patient cell line in amounts similar to a
control cell line (data not shown).
The 2q35 breakpoint interrupts the coding sequence of the
NHEJ1 gene. The breakpoint lies within intron 5 and interrupts
the coding sequence for the NHEJ1 protein after amino acid 196
(Fig. 3). We tested the expression of the SLC23A3 gene, a member
of the solute carrier family, which is located 0.5kb downstream
of NHEJ1. This gene is expressed in kidney but not in fetal
brain (Fig. 4A).
ATruncated NHEJ1Transcript Is Present
in the Patient’s Cells
To determine the amount of NHEJ1 transcripts present in the
cells of the translocation patient, quantitative real-time PCR was
performed with primers located before and after the translocation
breakpoint and with primers spanning the translocation break-
point (Fig. 5). The amount of NHEJ1 transcript containing exons
6 to 8 (i.e., after the translocation breakpoint) is reduced by 50%.
In contrast, the amount of transcript containing exons 1 to 5 is
similar to the amount present in control cells. These results
indicate that a truncated NHEJ1 transcript is expressed in the
Expression of NHEJ1in Human and Mouse Tissues
Northern blots prepared using human mRNA originating from
different brain regions were hybridized with a NHEJ1 probe and
revealed the presence of two transcripts (Fig. 4B). The stronger
hybridizing band was estimated to be about 2.2kb and the weaker
band about 1.6kb by comparison with RNA molecular weight
markers. We searched the NCBI database for NHEJ1 ESTs
and identified EST sequences containing two different putative
polyadenylation signals near a genuine poly(A) tail in the 30
sequence. These putative sites are located in exon 8 and are
separated by 600bp, a size consistent with the size of the two
transcripts. These observations were confirmed using 30rapid
amplification of cDNA ends (RACE) and sequencing (data not
RT-PCR experiments indicate that the NHEJ1 transcripts are
expressed in human fetal brain, lung, and kidney, with weaker
signals detected in other tissues (Fig. 4A). Northern blot analysis
using human nervous system tissues reveals that these transcripts
are detected primarily in cerebellum and cerebral cortex (Fig. 4B).
We also performed quantitative RT-PCR on total RNA from
mouse embryos at different developmental stages. The NHEJ1
transcript is highly expressed at embryonic day 12.5 (E12.5) with
the level of expression decreasing thereafter, suggesting regulated
expression during development (data not shown).
Expression of NHEJ1During Early Human
The expression of NHEJ1 during early human development was
examined by in situ hybridization in tissue sections (Fig. 6).
NHEJ1 is expressed extensively in the central nervous system
FIGURE 3. Schematic representation of the breakpoint regions. Chromosome 7 (top) and chromosome 2 (bottom) BAC contigs
showing the position of STSs in the critical region. In 7p22 the closest genes to the breakpoint are CARD11 and SDK1. In 2q35,
the breakpoint is located within intron 5 of NHEJ1. [Color ¢gure can be viewed in the online issue, which is available at www.
360 HUMAN MUTATION 28(4),356^364,2007
Human Mutation DOI 10.1002/humu
(CNS) from the earliest stage examined (Carnegie stage 16
[CS16] at about 37 days of development; Fig. 6A). At later stages
it is clear that expression is detected in the ventricular and
subventricular zones, i.e., in neuroblasts rather than in differ-
entiated neurons, and that there are regional differences in the
levels of expression detected (Fig. 6B and C). In the lateral
ventricles of the telencephalon, expression is relatively weak in the
medial wall, and stronger laterally, including the future cerebral
cortex. Signal is also detected in both lateral and medial ganglionic
eminences (Fig. 6C). In the diencephalon, strong expression is
detected in the dorsal thalamus and hypothalamus but expression
is much weaker in the ventral thalamus (Fig. 6B). Expression is
also strong in the developing cerebellum (Fig. 6B) but little or no
expression is seen elsewhere in the developing hindbrain (Fig. 6A
and C). NHEJ1 is detected in alar midbrain (data not shown) and
dorsal spinal cord (Fig. 6A and data not shown) and expression is
also present in non-CNS tissues including the developing tongue,
kidney, and digits (data not shown).
Mutation Screening in Patients With PMG
The fact that NHEJ1 is highly expressed in the embryonic
cerebral cortex and is disrupted in a fetus with PMG prompted us
to screen additional patients affected by PMG for mutations in
this gene. We therefore sequenced the coding region of NHEJ1 in
72 patients with perisylvian PMG, which is the most common
form, accounting for about 60% of patients [Leventer et al., 2001],
and 13 patients with PMG and heterotopia. No disease-causing
mutations were found. Other rare types of PMG that involve the
anterior frontal or occipital lobes have not yet been tested.
We report investigations of a fetus with a malformation of cortical
development consisting of extensive PMG and submicroscopic
nodular neuronal heterotopia throughout the white matter that was
found to have a de novo balanced translocation. One of the major
issues with this type of study is to determine if the observed phenotype
is a direct consequence of the presence of the chromosomal
rearrangement. Balanced translocations occur at a frequency of one
in 500 to 2000 live births and can also be identified in healthy
individuals [Baptista et al., 2005, Warburton, 1991]. However,
association of de novo balanced translocations and congenital
abnormalities was estimated to be a two-fold [Warburton, 1991] to
a six-fold increase [Bugge et al., 2000] compared to the general
population. This observation suggests that a causative link exists in
many cases between the interruption or modulation of gene expression
by a balanced rearrangement and the phenotype of its carrier.
To better understand the relationship between genotype and
phenotype in the patient that we studied, we mapped, cloned
and sequenced the two translocation breakpoints. We found that
the translocation is molecularly balanced with no significant loss or
gain of DNA. This is the most favorable situation, with a simple
exchange of genetic material between two chromosomes. More
interestingly, we showed that the NHEJ1 gene, also called
Cernunnos, XLF, or FJL12610 in two recent reports [Ahnesorg
et al., 2006; Buck et al., 2006], is the single transcription unit
disrupted by the translocation breakpoint in the cells of the
patient. Obviously, this finding makes it the best candidate to be
involved in the phenotype. Nonetheless, it was important to
consider the genomic context in the vicinity of the breakpoints
and to question the putative implication of the other genes located
in this area. On chromosome 7, the genes located the most closely
to the breakpoint are SDK1 and CARD11. SDK1 was shown to be
involved in synaptic adhesion during the development of the
retina [Yamagata et al., 2002] and in the pathogenesis of HIV-
associated nephropathy [Kaufman et al., 2004]. We cannot rule
out the possibility that this gene is implicated in the phenotype.
However, given its genomic location 100kb distal to the break-
point and its putative function, its involvement in the phenotype
of our patient is unlikely. We could not test if the transcription
of this gene was altered in the patient cells because SDK1 is not
expressed in lymphoblastoid cell lines. CARD11 has been classified
as a member of the membrane-associated guanylate kinase
(MAGUK) proteins with a caspase recruitment domain. It was
shown to be involved in the immune response with an implication
in NF-KappaB signaling [Bertin et al., 2001]. We have shown
that the two alleles of this gene are expressed in the patient
lymphoblastoid cell line, ruling out a major positional effect, at
least in this cell type. On chromosome 2, IHH (Indian Hedgehog)
is the best-characterized gene near the breakpoint. Mice deficient
for Ihh have abnormal skeletal morphogenesis [St-Jacques et al.,
1999]. Mutations in the human IHH gene result in brachydactyly
type A1 (MIM] 112500) and acrocapitofemoral dysplasia
(MIM] 607778). Also located on chromosome 2 is the human
SLC23A3 gene, which was not previously characterized other than
FIGURE 4. Expression of the genes present in the breakpoint
region. A: Expression pattern of the NHEJ1, SLC23A3,
CARD11, andSDK1genes using tissue-speci¢c RT-PCR.The gly-
ceraldehyde-3-phosphate dehydrogenase (GAPDH) control is
expressed in all tissues and was used as a control. B: Hybridiza-
pared with RNA isolated from di¡erent regions of the adult
HUMAN MUTATION 28(4),356^364,2007 361
Human Mutation DOI 10.1002/humu
its ability to encode a member of the large family of solute carrier
proteins. None of these genes appears to be an obvious candidate
for a neuronal migration disorder.
To further address the question of whether NHEJ1 was involved
in the phenotype of our patient, it was important to determine its
expression pattern since this has not been described before. Of
particular interest was the study of its expression during early
development of the human brain. We found that the expression
pattern of NHEJ1 is in good agreement with our hypothesis that
it could play a role in brain development. Indeed, we showed
that during early human development, the NHEJ1 transcript is
detected preferentially in the CNS including in regions that
subsequently give rise to the cerebral cortex. In the adult human
brain, the transcript is also abundant in the cerebral cortex and
in the cerebellum. More specifically, NHEJ1 is preferentially
expressed in the neocortical ventricular and subventricular zones
FIGURE 5. A truncatedNHEJ1transcript is present in the patient cells. A: Schematic representation of theNHEJ1gene showing the
positionof theprimersusedforthequantitativePCR. Amplicon 2 overlapsthebreakpointandampli¢esonlythenormalallelewhile
amplicons1and 3 can amplify putative 50or 30portions of thedisrupted allelein thepatient lymphocytes. B:Quantitative real-time
RT-PCR showing the relative expression of the di¡erent portions of NHEJ1 compared to b-actin in control and patient cells.The
expression levels of amplicons 2 and 3 were normalized and set at the same value than amplicon 1 to allow direct comparisons.
[Color ¢gurecan beviewedin theonlineissue,whichis available at www.interscience.wiley.com.]
FIGURE 6. ExpressionofNHEJ1duringearlyhumandevelopment. A: ParasagittalsectionofCS16 embryo (?37 daysofdevelopment)
showingexpressioninthedorsalmidbrain, hindbrain (developingcerebellum) andspinalcord. B:TransversesectionofCS21embryo
(?52 days of development) showing expression in the lateral ventricle, dorsal thalamus, ventral thalamus (weakly), and hypothala-
mus in the developing forebrain and the cerebellum in the developing hindbrain. C:Transverse section of CS23 embryo (?56 days
of development) showing expression in the lateral ventricles and ganglionic eminences of the developing forebrain.The expression
patterns were con¢rmed in two embryos at stages CS16,CS20, and CS21, and one embryo at CS23.Tissue in situ hybridizationwas
carried out as described in Materials and Methods using an NHEJ1 antisense probe. Corresponding sections were hybridized to an
NHEJ1senseprobeandnosignalwasdetected (data notshown). InpartsA, B, andC,thesignalisdetectedas ablue-purpledeposit.
c,cerebellum; d, dorsal thalamus; ge, ganglionic eminence; h, hypothalamus; H, hindbrain; L, lateral ventricle; M, midbrain;v,ven-
362 HUMAN MUTATION 28(4),356^364,2007
Human Mutation DOI 10.1002/humu
during periods of extensive neurogenesis. In contrast, there is little
or no expression in the cortical plate or intermediate zone. This
expression pattern suggests an expression in progenitor cells rather
than in postmitotic neurons and it is in good agreement with the
phenotype observed in the translocation patient consisting mainly
of a neurological phenotype. It is a similar pattern to that observed
for GPR56, a gene whose mutations cause frontoparietal bilateral
PMG, which is also highly expressed in neuronal progenitors [Piao
et al., 2004]. Since the proper cortical size and shape is determined
by the rate of production of neurons and glial cells in proliferative
zones [reviewed in Haydar et al., 1999] it is possible that an early
change in the expression of a gene strongly expressed in neuronal
precursors could alter subsequent patterning of the cerebral cortex.
Of the other sites of expression, both in CNS and non-CNS
tissues, the only one that appears directly relevant to the
phenotype is in the developing digits. The patient had syndactyly
of the fingers and toes. Considering this aspect of the phenotype,
the screening of NHEJ1 in patients with polymicrogyria associated
with limb defects deserves to be considered in the future.
Interestingly, the disruption of the coding region of NHEJ1 does
not lead to a simple reduction of its expression by 50%. The
portion of the transcript located 30to the breakpoint (exons 6 to 8)
on the translocated chromosome is no longer detectable in the
patient’s cells. In contrast, the 50region of the transcript located
upstream of the breakpoint (exons 1 to 5) is expressed in similar
amounts to the wild-type allele. This suggests that a truncated
transcript (possibly also containing a chromosome 7 transcribed
DNA fragment) is produced and is stable in the patient’s cells.
This truncated transcript encodes the NH2-terminal portion of
NHEJ1 containing the first 196 amino acids.
The recently described NHEJ1 mutations cause a phenotype
that differs significantly from the phenotype of our translocation
patient. In addition, the immunodeficiency and microcephaly
syndrome is autosomal recessive, whereas our patient has a wild-
type copy of the gene. If the disruption of NHEJ1 is responsible
for the observed phenotype, one explanation could be that the
expression of the truncated transcript causes a dominant negative
effect. The mutations identified in immunodeficient and micro-
cephalic patients are of various types with both missense and
nonsense mutations [Ahnesorg et al., 2006; Buck et al., 2006].
However, nonsense-mediated mRNA decay was not tested and it
is not known if these patients express an abnormal transcript or no
transcript at all. Indeed, one of the reported early truncating
mutations leads to a complete absence of NHEJ1 protein and an
unstable transcript [Ahnesorg et al., 2006]. This point is very
important since one may wonder whether or not the heterozygous
parents of the affected individuals are expressing an abnormal
but stable transcript from their mutated allele. If not, one of the
reasons why the translocation patient presents a different
phenotype could be the presence of a significant amount of
truncated NHEJ1 protein, especially because NHEJ1 is able to
homo- or heterodimerize. Truncated NHEJ1 proteins may be able
to sequester interactors such as XRCC4 into inactive dimers.
Whatever mechanism is true, it would be interesting to
investigate the functionality of the NHEJ pathway in the cells of
our patient or to perform MRI investigations in the immunodefi-
cient patients carrying truncating mutations to determine if they
do not suffer from subtle cortical disorganization.
We thank Regina Betz, Renzo Guerrini, and Daniela Pilz
for sending samples of patients. We thank Mareike Schnaars for
her help during the initial phases of this work. The human
embryonic tissue was provided by the Joint Medical Research
Resource at Institute of Human Genetics (IHG), Newcastle upon
Tyne (www.hdbr.org). V.C. is the recipient of a Ministe `re de la
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