Homozygous silencing of T-box
transcription factor EOMES leads
to microcephaly with
polymicrogyria and corpus
Lekbir Baala1,2, Sylvain Briault3,8, Heather C Etchevers2,
Fre ´de ´ric Laumonnier3, Abdelhafid Natiq1, Jeanne Amiel2,
Nathalie Boddaert4, Capucine Picard5, Aziza Sbiti1,
Abdellah Asermouh6, Tania Attie ´-Bitach2,7,
Fe ´re ´chte ´ Encha-Razavi2,7, Arnold Munnich2,7,
Abdelaziz Sefiani1& Stanislas Lyonnet2,7
Neural progenitor proliferation and migration influence brain
size during neurogenesis. We report an autosomal recessive
microcephaly syndrome cosegregating with a homozygous
balanced translocation between chromosomes 3p and 10q,
and we show that a position effect at the breakpoint on
chromosome 3 silences the eomesodermin transcript (EOMES),
also known as T-box-brain2 (TBR2). Together with the
expression pattern of EOMES in the developing human brain,
our data suggest that EOMES is involved in neuronal division
and/or migration. Thus, mutations in genes encoding not only
mitotic and apoptotic proteins but also transcription factors
may be responsible for malformative microcephaly syndromes.
Microcephaly syndromes are a heterogeneous group of genetic dis-
orders in which affected individuals have a head circumference below
3 s.d., a small and malformed brain and cognitive deficiency. A
large consanguineous Moroccan family was referred to us with a
marked prenatal-onset microcephaly (mean occipitofrontal circum-
ference at birth ¼ –4 s.d.) and severe motor delay with hypotonia in
four affected children. Early lethality was observed in three children
(death at 15–18 months of age) due to respiratory distress after
chronic infections. The surviving child (patient V.6) has had a
persistent fever since birth and recurrent infections (Supplementary
Notably, in addition to congenital microcephaly, these individuals
consistently showed corpus callosum agenesis, bilateral polymicro-
gyria, ventricular dilatation and a small cerebellum, as demonstrated
by brain CT and MRI scans (Supplementary Fig. 1 online).
This defines a congenital microcephaly with extensive poly-
microgyria1, the mechanisms of which are not known and are
presumably ascribed to abnormal neuronal and/or glial proliferation
or apoptosis. This condition is clearly different from primary micro-
Despite a pattern of affected individuals in the pedigree suggestive
of an autosomal mode of inheritance, chromosomal analysis on
leukocytes showed a reciprocal balanced translocation between chro-
mosomes 3p and 10q segregating in the family (Fig. 1a). We found
that the translocation was homozygous in each of the four affected
individuals studied (46,XY,t(3;10)(p24;q23)2x), whereas healthy
parents were heterozygous. Genome-wide comparative genomic
hybridization pattern analysis showed that chromosomal rearrange-
ment had occurred without detectable loss or gain of genetic material
at a resolution of 3 Mb (data not shown).
Linkage analysis between the disease trait and polymorphic markers
of chromosomes 3p and 10q defined two regions identical by descent
of 27 Mb and 11.4 Mb, respectively (Fig. 1b). The two-point lod score
between the translocation breakpoint used as a marker, and the disease
locus peaked at significant values of 3.45. In addition, we excluded
linkage to the six known primary microcephaly loci (MCPH1 to
MCPH6) (Supplementary Methods online). One hypothesis is that a
locus involved in neuronal proliferation and/or migration maps to
either chromosome 3 or chromosome 10 and that the translocation
breakpoint disrupts the disease-causing gene.
We therefore established a physical map of chromosomal regions
3p24 and 10q23 and characterized the BACs that encompassed the
breakpoints for each chromosome (BAC RP11-9a14 and RP11-
102H24 on chromosomes 3 and 10, respectively; Supplementary
Fig. 2 and Supplementary Table 1 online). Furthermore, DNA
sequencing of long-range PCR products allowed us to characterize
the translocation breakpoints and demonstrate that they occurred
without any deletion at nucleotide positions 27954024 and 82932753
(NCBI build 36.1) on chromosomes 3p and 10q, respectively (Sup-
plementary Methods and Supplementary Fig. 3 online). Notably,
neither of the translocation breakpoints disrupted a known or pre-
dicted gene coding sequence on either chromosome, suggesting that
the translocation affected surrounding gene(s) by a positional effect.
Among the nine annotated genes located closest to the breakpoints,
EOMES (MIM 604615) was considered the best candidate gene.
EOMES encodes a transcription factor, a member of the T-box family,
that is critical in vertebrate embryonic development of the central
nervous system and mesoderm2–4. The EOMES locus (NCBI build
36.1, position 27732891–27738789) is located 215 kb 3¢ to the
translocation breakpoint on chromosome 3p and is transcribed
away from it. We sequenced the six annotated coding exons of
EOMES in affected family members and did not find any mutation
in these or in a 5¢ predicted noncoding exon that is located 136 bp
Received 27 November 2006; accepted 1 February 2007; published online 11 March 2007; doi:10.1038/ng1993
1De ´partement de Ge ´ne ´tique Me ´dicale, Institut National d’Hygie `ne, Rabat, Maroc.2INSERM U781, Ho ˆpital Necker, De ´partement de Ge ´ne ´tique, Paris, France.
3INSERM U619, Faculte ´ de Me ´decine, Tours, France.4Ho ˆpital Necker, Service de Radiologie Pe ´diatrique and5Centre d’e ´tude des de ´ficits immunitaires, Paris, France.
6Ho ˆpital d’Enfants CHU Avicenne, Rabat, Maroc.7Universite ´ Re ´ne ´ Descartes - Paris 5, Paris, France.8Present address: Laboratoire de ge ´ne ´tique, CHR La Source,
Orle ´ans, France. Correspondence should be addressed to S.L. (firstname.lastname@example.org).
454 VOLUME 39 [ NUMBER 4 [ APRIL 2007 NATURE GENETICS
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5¢ to the transcription start site and may be used in several alternative
EOMES transcripts (Supplementary Table 2 online).
However, we identified a synonymous SNP in EOMES exon 6 (A/G,
rs6783101; estimated frequency of the rare allele A in the African
American population ¼ 5.9%). The A allele of SNP rs6783101
cosegregated with the derivative 3p–10q translocated chromosome
in the family, allowing its use as a cis marker to test allele-specific
expression at the EOMES locus in cell lines. Indeed, quantitative
RT-PCR showed no EOMES expression in affected individuals
(Fig. 2a), whereas the only transcribed allele in a heterozygous parent
carried the wild-type G nucleotide at SNP rs6783101 (Fig. 2b),
demonstrating monoallelic expression and specific silencing of the
EOMES allele on the translocated chromosome. Qualitative RT-PCR
showed that, among the nine genes proximate to the breakpoints, the
mRNA transcribed from EOMES on chromosome 3p24 was the only
one absent from lymphoblast cell lines from affected individuals
(Fig. 1b, Supplementary Methods and Supplementary Table 2).
Tbr2 has recently been shown to be pivotal in the developing mouse
neocortex, along with other transcription factors, including Pax6,
NeuroD and Tbr1 (refs. 5,6). Therefore, we investigated the expression
pattern of EOMES in human prenatal tissues at different stages of
development (Fig. 2c–i and Supplementary Methods). The EOMES
transcript was visibly expressed at 7 weeks of development (Carnegie
stage 19) in a pattern apparently restricted to the forebrain floorplate
of the central nervous system (Fig. 2d). However, we observed distinct
EOMES expression within the mantle layer (Fig. 2h) and migrating
neuroblasts (Fig. 2i) of the telencephalon at 12.5 weeks. This limited
expression pattern differs from that of the mouse, implying
evolutionary divergence of noncoding control elements, as shown
for brain-specific expression of WNT7A7. This pattern supports a role
for human EOMES in late neuronal development and suggests that its
silencing contributes to the disease phenotype in individuals with
Proliferation and neuronal fate specification are key events in the
developing ventricular zone and subventricular zone (SVZ) of the
central nervous system5,6. Mouse Eomes (Tbr2) is expressed in these
sites and may be involved in precursor proliferation. In humans,
neuronal migration occurs largely between the 12thand 24thweek of
gestation. This period, preceded and accompanied by intense cell
division in the ventricular zone, corresponds to the time frame in
which we observe EOMES expression in the telencephalon. During
development, a number of other transcriptional regulators balance
cortical cell proliferation and differentiation8. In the cortex, radial glia
produce both neurons and glia9, whereas intermediate progenitor cells
produce only neurons and divide away from the ventricular surface.
The transition from radial glia to intermediate progenitor cell in the
mouse is associated with upregulation of Eomes and downregulation
of Pax6, whereas the subsequent transition from intermediate pro-
genitor cell to postmitotic neuron is marked by downregulation of
Eomes and upregulation of Tbr1 (ref. 5). Interruption of the efficiency
of precursor production, or secondary impairment of neuronal
migration, would be predicted to result in a smaller telencephalic
Figure 1 Segregation, genetic and fine physical mapping of the disease locus. (a) Simplified family pedigree. Chromosomal and molecular analyses were
carried out on the core family (indicated by a triangle) after we obtained their informed, written consent, under supervision by the Necker Hospital ethics
committee. Filled black symbols indicate individuals with the microcephaly syndrome. R banding of chromosomes 3 and 10 is schematically presented;
arrowheads indicate translocation breakpoints. (b) Mapping of the disease locus using homozygosity for a balanced reciprocal translocation, and expression
pattern in surrounding candidate genes. The BACs crossing the breakpoints were identified for both chromosomes 3p and 10q (shaded boxes). The genetic
map shows the region cosegregating with the disease phenotype (filled black bars). Candidate genes are indicated on the physical map and in Supplementary
Table 2. Results of qualitative PCR on lymphoblast cDNA from individual V.6 are shown at right (+, expressed; –, not expressed).
NATURE GENETICS VOLUME 39 [ NUMBER 4 [ APRIL 2007455
© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
surface. However, the fact that normally sized brains are also asso-
ciated with corpus callosum agenesis, as well as the complexity of
corpus callosum development, does not permit the conclusion that the
absence of the corpus callosum in these individuals is readily explained
by a neuronal migration or proliferation defect alone. Furthermore,
EOMES may have a species-specific role in corpus callosum develop-
ment. We screened six unrelated individuals with absent corpus
callosum as a primary feature for coding sequence mutations of the
EOMES gene and did not find any mutations or rearrangements (data
not shown). At least 18 numerical or structural chromosomal aberra-
tions have been reported in individuals with corpus callosum defects,
and more heterogeneity is likely1.
Eomes has been described in mouse as a key transcription factor for
memory CD8+T cells and for full effector differentiation of CD8+
T cells10. Eomes is induced in effector CD8+T cells after viral infection
and after expression increases in memory T cells; it is induced in
memory cells only after bacterial infections leading to high levels of
interleukin 12, which favors the acute host response11. However, we
did not detect any major immune deficiency and/or quantitative
abnormalities in the T CD8 subset in affected individual V.6 (Supple-
mentary Note). Further T functional studies must be performed to
explore the effect of EOMES silencing on the immune system.
The genetic and expression evidence that we provide supports the
conclusion that homozygous silencing of the human EOMES locus
results in a microcephaly syndrome with polymicrogyria and agenesis
of the corpus callosum. Unusually, silencing of the EOMES locus in
the individuals studied is ascribed to a position effect resulting from a
translocation breakpoint. As no additional EOMES exons have been
detected (Supplementary Methods) and the transcriptional direction
of EOMES gene is away from the breakpoint on the native chromo-
some 3p, we hypothesize that a cis-regulatory sequence12lying 215 kb
or more 5¢ to the EOMES locus may have been separated from the
EOMES core promoter.
Because the full knockout of Tbr2 in mice leads to embryonic
lethality before implantation4,13, the essential role of this gene product
in brain development has not been hitherto emphasized. The motor
delay with hypotonia observed in the individuals with microcephaly
syndrome in our study, as well as their reduced cerebellar size, recalls
the recently demonstrated co-opting of this developmental gene cascade
in the mouse cerebellum, in which precursors of the neurons of the
deep cerebellar nuclei both express and require Eomes transcripts14. We
did not observe any EOMES expression in the developing human
cerebellum at Carnegie stage 19 or 12.5 weeks of development; later
stages were not accessible for analysis. Although other T-box family
member genes (TBX3 and TBX5) have been reported to be involved in
human developmental disorders involving the heart and skeleton
(ulnar-mammary (MIM181450) and Holt-Oram (MIM142900) syn-
dromes, respectively), our report is the first to implicate EOMES in a
severe neurological malformation in humans.
Note: Supplementary information is available on the Nature Genetics website.
The authors thank the microcephaly syndrome family for their participation.
We thank Z. Al-Houssaini, N. Bahi-Buisson, C. Chirol, M. Cle ´ment-Ziza,
N. Moussok, A. Pelet, S. Romana, C. Schatz and M. Vekemans for their
assistance. This study was funded by INSERM, Agence Nationale de la Recherche
and the Fondation pour le Recherche Me ´dicale.
COMPETING INTERESTS STATEMENT
The authors declare no competing financial interests.
Published online at http://www.nature.com/naturegenetics
Reprints and permissions information is available online at http://npg.nature.com/
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Figure 2 Silencing of the translocated EOMES locus and normal EOMES expression in the developing human brain. (a) Quantitative PCR of EOMES exon
6 on cDNA derived from lymphoblast lines of a control individual, the homozygous affected individual V.6 and his heterozygous father, normalized to the
expression level of b-actin. Bars represent s.d. from three replicates. (b) Monoallelic expression of the wild-type EOMES locus. Genomic sequence traces
centered on the A/G SNP found in EOMES exon 6 are shown for affected individual V.6, his father and a control. RT-PCR sequences of the EOMES mRNA in
the same individuals are shown below. We did not detect any EOMES mRNA in V.6, whereas only the non-translocated EOMES allele was expressed in the
heterozygous father. (c–i) Parasagittal sections through the head of a Carnegie stage 19 embryo (7 weeks of development). c, hematoxylin-eosin (HE) stain.
d, enlargement of basal forebrain showing discrete EOMES expression in the floorplate (arrowhead) as compared with the adjacent sense control hybridized
slide (e). d and e are 75–80 mm medial to c. We observed localized EOMES expression when we hybridized an antisense probe (f) versus a sense probe (g)
in adjacent frontal sections through the telencephalon of a fetus at 12.5 weeks of development. Magnifications of the cortical mantle layer (h), with intense
signal in the subventricular zone, and dense neuroblasts (i) of the future basal ganglia.
456 VOLUME 39 [ NUMBER 4 [ APRIL 2007 NATURE GENETICS
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Supplementary figure 1: The following features were observed in all patients : i) extreme microcephaly (V.3, V.5 and
V.6), ii) craniosynostosis secondary to microcephaly (a, b, d, e), iii) corpus callosum agenesis (CT scan view (c) and MRI
view (g, h), iv) other brain malformations, namely: bilateral cortical polymicrogyria (h white arrow), myelinization delay
on anterior arm of internal capsulae (i white arrow), a reduced cerebellum without malformation of posterior fossa (g), v)
Individual V.5 also presented with a left kidney pyelo-ureteral junction abnormality (f). The photographs are published
with parental consent.
Supplementary Figure 2
FISH analyses of BACs (Bacterial artificial chromosome) RP11-9A14 on chromosome 3p (A) and
RP11-102H24 on chromosome 10q (B), encompassing the breakpoints (green probes indicated by
white arrows). The specific chromosome 10 satellite probe is a purple/red colour (red arrows).
der3 : derivative chromosome 3; der10 : derivative chromosome 10.
Supplementary figure 3 : Sequencing of the junction fragments on 3p24 (blue arrow)
and 10q22 (green arrow) demonstrated that no deletion was involved. A 56 nucleotide
sequence originating from a Long Interspersed Nuclear Element (LINE) repeat was
intercalated at the translocation breakpoint (red dotted line).
LINE sequence Chromosome 3
Supplementary Table 1: BAC probes encompassing the 3p and 10q translocation breakpoint. All BAC clones
are from the RP11 library. The probes overlapping the breakpoints are in bold case (see Supplementary Figure
Chromosome 3 Chromosome 10
Probes Accession N° Physical
Probes Accession N° Physical
Supplementary Table 2: Methods for RT-PCR analysis of candidate genes on chromosomes 3p and 10q. In addition,
the PRO-NRG3, DC-TM4F2, and EOMES genes were fully sequenced.
Genes & accession N°
Primer sequences and exon location Physical Position (UCSC)
Solute carrier family 4
T box brain 2 /
5’-ACGAGGCTTTGGAGAAGCTCTTT, Exon 15
5’-ACACCAAAAGAGCACATCTGGAAT, Exon 17
5’-GAACCTGGCTTCGCTAACG, Exon 1F
5-azacytidine induced 2
Modified Nov 1, 2006 to
5’-GAAGAACAGTTGCGCCGGTCC, Exon 4-5F
5’-GGTGTTCCTCTCGAAGCCAG, Exon 6R
Discontinued from RefSeq
superfamily member 14)
Blood samples were collected from all four patients and their parents. Informed written consent was obtained
from the families. Cytogenetic analysis was performed using standard R-band techniques. FISH experiments, using
BACs as probes (CHORI Center), were performed on metaphases of patient IV.5 and his father IV.7 (Fig. 1). Total
DNA from these BACs was labelled with biotin by nick-translation as previously described. The probes were revealed
by using avidin-FITC (Sigma). All slides were counter-stained with DAPI (Sigma). The slides were observed under a
Zeiss epifluorescence microscope (Axiophot) connected to the Powergene 810 probe system (Perspective Scientific
International LTD). After the CGH experiment was performed, the slides were visualized on an epifluorescent
microscope (DMRXA, Leica Microsystems). Images were processed and analyzed with the Quips CGH software (Vysis
- Chromosomes 3p and 10q
Linkage analysis was performed in chromosome 3p and 10q to identify a homozygous region by descent for
each of the two chromosomes which prompted us to select BACs for the physical fine mapping of the breakpoint. The
following microsatellite markers were used: D3S1304, D3S1263, D3S1259, D3S1286, D3S1293, D3S1266, D3S1582,
D3S1613, D3S3717, D3S3721; and D10S556, D10S195, D10S201, D10S1686, D10S1744, D10S198, D10S192,
When assuming a fully penetrant autosomal recessive disorder, linkage analysis between the disease phenotype
and translocation breakpoints used as polymorphic markers yielded a maximum lodscore of Z = 3.45 at 3p24 or 10q23,
when including three affected individuals only. If one common ancestor carried the translocation, the likelihood that
three affected offspring would be homozygous for the translocation by chance is 1/4,000.
- Exclusion of MCPH loci
We did homozygosity mapping and linkage analysis by using 36 microsatellite markers across the six MCPH
loci from the family branch with two affected individuals and one healthy sister. We excluded the following loci:
MCPH1 (D8S1798, D8S277); MCPH2 (D19S414, D19S570, D19S220, D19S881, D19S417, D19S223, D19S197,
D19S198, D19S423, D19S420, D19S900); MCPH3 (D9S1872, D9S1682); MCPH4 (D15S1007, D15S1042,
D15S1012, D15S1044, D15S994, D15S968, D15S1006, D15S978, D15S126, D15S982, D15S1003, D15S117,
D15S964, D15S643, D15S155); MCPH5 (D1S238, D1S422, CRB1 [a CA repeat marker designed locally, primers
available on request] and D1S413); MCPH6 (D13S742, D13S221).
DNA sequence analysis
DNA was extracted from peripheral blood (or lymphoblastic cell lines) according to standard protocols. We
analysed candidate genes by genomic and/or cDNA sequencing. PCR products were purified and directly sequenced in
both directions on an ABI PRISM 3130 DNA sequencer (Perkin Elmer-Applied Biosystems) using the dye terminator
method according to the manufacturer’s instructions (cf. Nature Protocols).
- Total RNA extraction and RT-PCR analysis
Total RNA was extracted from cultured lymphoblastic cell lines using the RNeasy Mini kit (Qiagen) according
to manufacturer’s instructions. A sample of extracted RNA was electrophoresed on a 2% agarose gel to verify integrity.
First strand cDNA was synthesized using random hexamer primers (GeneAmp RNA PCR kit, Applied Biosystems).
Reverse transcription was carried out at 42° for 15 min, at 99° for 5 min, and at 5° for 5 min. RT-PCR was performed
using gene-specific primers (Supplementary Table 2).
- Quantitative RT-PCR :
Quantitative measurements of changes in gene expression were performed using a LightCycler (Roche
Diagnostics, Indianapolis, Ind.) thermocycler. Q-PCR was performed (operating system version 3.0) in 10µl mixtures
containing 1 µl of Faststart DNA Sybr Green I (Roche Molecular Biochemicals), 1.5 mM MgCl2, 0.5 mM each primer
(EOMES-Ex6F2-5’-GGACTACCATGGACCTCCAGAACA-3’, and EOMES-Ex6R1 5’-
TCTTCAGCATTAATGTCCTCACACTT-3’), and 5 µl of extracted DNA (1 to 25 ng). The reaction was performed
with preliminary denaturation for 10 min at 95°C (slope, 20°C/s), followed by 40 cycles of denaturation at 94°C for 10 s
(slope, 20°C/s), annealing at 60°C for 5 s (slope, 20°C/s), primer extension at 72°C for 8 s (slope 20°C/s), and product
detection at 77°C for 5 s (slope, 20°C/s). A final cooling step was performed at 4°C for 1 min (slope, 20°C/s). A 239-bp
product resulted from the reaction. Experiments were repeated in triplicate and normalized for input cDNA against beta-
- RACE (Rapid Amplification of cDNA Ends)
We performed 3’ RACE analysis using the GeneRacerTM Kit (Invitrogen). The first-strand of cDNA was
amplified using a forward gene specific primer within the published first exon (Forward GSP: 5’-
TCCGAGCGGTACTACCTCCAGT) and the GeneRacerTM Oligo dT Primer according to manufacturer’s instructions.
We did not find supplementary exons 3’ to the six published exons of EOMES.
- In situ hybridisation
Normal human embryos and foetal tissues were obtained after elective termination of pregnancy in agreement
with current French bioethical legislation (94-654 and 00-800), the Necker Hospital CCPPRB and National Ethics
Committee recommendations (N° 1 of May 22, 1984). Embryonic stages were established according to Carnegie
staging (CS) classification. Six different embryonic stages (CS8 (d18), CS9 (d20), CS15 (d33), CS19 (d 47–48)) as well
as two fetal stages (14.5 and 24 weeks) were studied. Tissues were fixed in 4% phosphate buffered paraformaldehyde,
dehydrated, and embedded in paraffin blocks. Five micron thick serial sections were cut. Exon 2 primers were selected
for PCR amplification (F: 5’-CCTGTTCTAGGACATCCCAATT -3’ R: 5’-GAGGGTTACGATTTCTTC-3’). A T7
promoter sequence extension (TAATACGACTCACTATAGGGAGA) was added at the 5’ end of each primer. T7F/ R
and F/ T7R primers allowed the amplification of sense and antisense templates respectively, specific to the EOMES
gene. Riboprobe labelling with 35S-UTP, tissue fixation, hybridization, and photographic development were carried out
according to standard protocols as previously described.
Supplementary note Download full-text
During the first week of life, he had a fever (38.6-40.3°C) that was unresponsive to
antipyretics, caused by Escherichia coli sepsis. Biological explorations showed augmented
leucocytes (16 780 /µl) and lymphocytes (61.7 %; Normal: 19 - 48 %), diminished levels of
red blood cells, haemoglobin, hematocrit , and polymorphonuclear neutrophils. At the age of
3 months, he presented an urinary infection (pyelonephritis) associated with fever and
Klebsiella pneumoniae isolated in the urine.
The cytobacterial investigation in the cerebrospinal liquid showed a very high level of
red blood cells (120/µl; N: 1 - 2) and leucocytes (4/µl; N: <3). The direct bacteriological exam
revealed absence of germs.
The haematological survey was essentially normal except the polymorphonuclear
basophils which were two fold higher than normal. At the time of publication, the infant at 9
months of age has had a fever on every measurement and subsequent episodes of infections.
The exploration of lymphocyte subtypes of patient V.6 had found normal distribution of T, B
and NK cells. For T CD8 subtypes the percentage of memory and naive cells was normal, but
the patient presented a slight increase in effector T (CD8) cells with normal expression of
The results of all other standard immunological explorations were normal, including
serum immunoglobulin levels (at the age of 6 and 9 months), antibody responses to proteins
and complement (CH50, C3, C4).