Clin Genet 2008: 74: 392–395
Printed in Singapore. All rights reserved
# 2008 The Authors
Journal compilation # 2008 Blackwell Munksgaard
CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2008.01078.x
Short Report
Confirmation of RAX gene involvement in
human anophthalmia
Lequeux L, Rio M, Vigouroux A, Titeux M, Etchevers H, Malecaze F,
Chassaing N, Calvas P. Confirmation of RAX gene involvement in
human anophthalmia.
Clin Genet 2008: 74: 392–395. # Blackwell Munksgaard, 2008
Microphthalmia and anophthalmia are at the severe end of the spectrum
of abnormalities in ocular development. Mutations in several genes have
been involved in syndromic and non-syndromic anophthalmia.
Previously, RAX recessive mutations were implicated in a single patient
with right anophthalmia, left microphthalmia and sclerocornea. In this
study, we report the findings of novel compound heterozygous RAX
mutations in a child with bilateral anophthalmia. Both mutations are
located in exon 3. c.664delT is a frameshifting deletion predicted to
introduce a premature stop codon (p.Ser222ArgfsX62), and c.909C.G is
a nonsense mutation with similar consequences (p.Tyr303X). This is the
second report of a patient with anophthalmia caused by RAX mutations.
These findings confirm that RAX plays a major role in the early stages of
eye development and is involved in human anophthalmia.
L Lequeuxa,b, M Rioc,
A Vigourouxa,b, M Titeuxa,
H Etcheversa, F Malecazea,d,e,
N Chassainga,b,d and
P Calvasa,b,d
aINSERM, U563, Centre de
Physiopathologie de Toulouse Purpan,
Toulouse, France, bCHU Toulouse,
Hoˆpital Purpan, Service de Ge´ne´tique
Me´dicale, Toulouse, France,
cDe´partement de Ge´ne´tique Me´dicale,
Hoˆpital Necker-Enfants Malades, Paris,
France, dUniversite´ Toulouse III Paul-
Sabatier, Toulouse, France and eCHU
Toulouse, Hoˆpital Purpan, Service
d’Ophtalmologie, Toulouse, France
Key words: anophthalmia –
microphthalmia – OAR transactivation
domain – RAX
Corresponding author: Professor Patrick
Calvas, Service de Ge´ne´tique Me´dicale,
Pavillon Lefebvre, CHU Purpan, Place du
Dr Baylac, 31059 Toulouse Cedex 9,
France.
Tel.: 133 5 61 77 90 79;
fax: 133 5 61 77 90 73;
e-mail: calvas.p@chu-toulouse.fr
Received 27 March 2008, revised and
accepted for publication 3 July 2008
Microphthalmia and anophthalmia are at the
severe end of the spectrum of abnormalities in ocu-
lar development. The combined occurrence rate for
these two malformations is 1/10,000 births (1, 2).
Mutations in several genes have been isolated in
syndromic and non-syndromic anophthalmia. Het-
erozygous mutations in SOX2 account for approx-
imately 10% of anophthalmia (3, 4). Other genes
have been identified as causing anophthalmia or
extreme microphthalmia in humans (PAX6,
OTX2, CHX10, STRA6, and BMP4) (5, 6). These
latter genes are implicated in a very small propor-
tion of affected individuals, implying wide genetic
heterogeneity to match the phenotypic variability.
The RAX homeobox gene is essential for verte-
brate eye development. RAX transcription begins
in the anterior neural plate and then simulta-
neously in the eye field and in the ventral forebrain
(7). Even before PAX6, its expression is critical to
defining the eye field during early development in
animal models (8). The lack of RAX expression
hampers optic vesicle formation and leads to brain
size reduction in mouse, while ectopic expression
induces the appearance and proliferation of reti-
nal pigment epithelium cells in Xenopus (9). The
function of the RAX gene in eye development is
yet not fully understood, but there is additional
evidence from animal studies that it is involved
in the proliferation of neural and retinal cells
(10). In humans, the role ofRAX in eye formation
is clearly supported by the association of anoph-
thalmia and sclerocornea in a patient bearing
392

a truncating mutation and a missense mutation,
both located in the DNA-binding helix of the ho-
meodomain and reducing the DNA-binding abil-
ity of the resulting protein (11). We report in this
study the case of a new patient with bilateral
anophthalmia associated with two distinct and
novel truncating mutations of the RAX gene.
Patient, materials and methods
Patient
The proband, a 2-year-old girl, is the third child
born to non-consanguineous, healthy Algerian
parents. There was no relevant familial history
of ocular malformation or remarkable disease.
The pregnancy was uneventful, and the prenatal
ultrasonography was not suggestive of anomaly.
Delivery occurred at 41 weeks of amenorrhea
without neonatal difficulties. Birth weight was
3200 g. At birth, bilateral small palpebral fissures
were noted without other malformation or dys-
morphic features. Anophthalmia was subsequently
confirmed. Psychomotor development was within
the normal range with head held up at 3 months,
sitting at 10 months, and walking at 1 year.
Speech developed normally. A slight growth
defect was recorded at 14 months, with weight at
20.5 standard deviation (DS) (9020 g), height at
21 DS (72 cm) and head circumference at22 DS
(44 cm). Abdominal and pelvic ultrasonography
detected no visceral anomalies. Orbital and cra-
nial magnetic resonance imaging scan showed
bilateral absence of eyes with only fibrous tissue
in the orbits (Fig. 1). Optic nerves and chiasma
were hypoplastic. Extraocular muscles appeared
to be relatively preserved. The hypothalamus
and pituitary glandwere normal. No cerebralmal-
formation was observed.
Molecular analysis
Parents gave their informed consent, according to
French law, to participate in this study. DNAwas
isolated by standard procedures from peripheral
white blood cells of the proband. Routine exami-
nation ruled out rearrangements or point muta-
tions of SOX2 and PAX6 genes. The three RAX
exons, with exon–intron borders, were amplified
by polymerase chain reaction (PCR) using previ-
ously published primers (11). PCR fragments were
subsequently purified with QIAquick Gel Extrac-
tion Kit (QIAGEN SA, Courtaboeuf, France),
and both strands were sequenced using Big
Dye DNA sequencing kit (Applied Biosystems,
Warrington, UK). Reactions were analyzed in
an ABI 3100 sequencer (Applied Biosystems).
Sequence variations were numbered considering
adenine of the ATG initiation codon as the
first nucleotide (GenBank accession no. NM_
013435.2). The changes were verified by perform-
ing independent PCRand sequencing reactions on
the proband’s DNA.
Exon 3 of the RAX gene was PCR amplified
from the patient’s DNA as above (11). The result-
ing 602-bp fragments were cloned into the pGEM-
T vector (Promega, Charbonnie`res, France).
JM109 competent cells were transformed and
grown on Luria-Bertani agar plates. DNAs from
10 expanded LacZ-deficient clones were extracted
using Promega Wizard miniprep purification sys-
tem. Further sequencing was performed using the
ABI-Big Dye terminator 3.1 on an ABI 3100
sequencer (Applied Biosystems).
Results
Sequence analysis of the proband’s DNA revealed
two novel mutations, both located in exon 3 of the
RAX gene. c.664delT frameshifting deletion gen-
erates a premature stop codon (p.Ser222-
ArgfsX62). c.909C.G is a nonsense mutation
changing a tyrosine at position 303 to a stop
codon (p.Tyr303X). These mutations were not
found in a panel of 96 control chromosomes. Both
are predicted to lead to a truncated protein so that,
if not submitted to nonsense-mediated mRNA
decay, the predicted RAX proteins lack the puta-
tive otp, aristaless, rax (OAR) transactivation
domain and are non-functional (7).
Fig. 1. Magnetic resonance imaging scan of the proband.
Note absence of ocular structures replaced by fibrous tissue.
RAX mutations in anophthalmia
393

As this family left the country, DNA from the
proband’s parents was unavailable, and thus, seg-
regation analysis of these two mutations was
impossible. Nevertheless, the c.664delT and the
c.909C.G mutations were shown to lie in trans
after sequencing of the cloned products of the pa-
tient’s RAX exon 3 (Fig. 2).
Discussion
This is the second report of human anophthalmia-
associated mutations of the RAX homeobox gene
(11). While the parents were not carefully exam-
ined, they did not complain of any visual impair-
ment at the time their child was evaluated. The
probandwas demonstrated to bear composite het-
erozygous mutations on both alleles of the RAX
gene. The parents are thus likely to each be healthy
carriers of a heterozygous mutation, unless one of
these mutations appeared de novo. This would
confirm the recessive inheritance of RAX muta-
tions in ocular dysgenesis.
The phenotype, reported in this study, consist-
ing in bilateral and symmetric anophthalmia is
more severe than the one previously described.
This first patient had right anophthalmia and left
microphthalmia and sclerocornea (11). One of the
causative mutations (p.Gln147X) induced, as pre-
dicted for the two mutations reported in this
study, a truncation of the protein. The other was
a missense p.Arg192Gln, with a milder effect on
the protein, which conserved a low activity. This
could suggest that the observed phenotypic vari-
ability be correlated with the mutation severity.
However, definite conclusions cannot be drawn
in view of the limited number of observations.
In animal models, all truncating mutations have
been reported to have severe effects and lead to the
absence of eye development (9, 12, 13). In con-
trast, antisense or morpholino inhibition in Xen-
opus acts in a dose-dependant manner, leading to
graduated phenotypes ranging from eye reduction
to anophthalmia (14). In this report, the location
of the mutations in the last exon makes nonsense-
mediated mRNA decay unlikely (15). This is in
accordance with the observation that, in the cel-
lular model used by Voronina et al (11), the more
proximal p.Gln147X mutation allowed transla-
tion of a large amount of protein. These facts sug-
gest that the twomutations we report in this study
lead to truncated proteins, both lacking the C-
terminal part containing the critical OAR funct-
ional domain (7). Absence of RAX C-terminus is
known to abolish its proliferative effect in Xeno-
pus (14). Furthermore, regulation of transcrip-
tional activity of several other homeobox genes
by the OAR domain has been suggested in other
studies (7, 16, 17). Thus, p.Ser222ArgfsX62 and
p.Tyr303X are thought to drastically impair RAX
target genes expression. The precise delineation of
the mechanistic effects of these mutations must
therefore await binding studies, and an important
goal for future research will be the identification
of the putative genes that can modulate RAX
activity through direct interaction.
To date, no cerebral malformation has been
associated with RAX mutations in man. This is
Fig. 2. Electropherograms showing the two mutations on RAX exon 3 (a and d) in comparison with wild-type sequence (c and
f). Sequencing of cloned patient’s exon 3 amplimers in a pGEM-T vector (b and e) demonstrated that mutations were not
located on the same alleles. Mutated codons are underlined.
Lequeux et al.
394

surprising in the light of the observations in insect,
batracian, fish and rodent models, where RAX
consistently participates in brain development
and homozygous null alleles cause severe cerebral
malformations (9, 14, 18, 19). A similar situation
is seen, however, with respect to theHesx1 homeo-
box-containing transcription factor, which in
mice has a similar early role and an overlapping
domain to that of Rax but is downstream of Pax6
and Otx2 (20) and Rax itself (21). While Hesx1
mouse mutants can demonstrate anophthalmia in
addition to cerebral anomalies, human patients
have either isolated pituitary malformations or
septo-ocular dysplasia, with no further retinal
involvement (22). In a complementary fashion
and unlike SOX2 or OTX2 mutations, no extra-
ocular malformations have been observed inRAX
ocular dysgenesis patients. The patient reported
previously by Voronina et al. (11) was diagnosed
as autistic. The patient reported in this study
seems to have normal psychomotor development,
although she is too young to exclude the possibil-
ity of developmental delay and/or autistic fea-
tures. Thus, RAX phenotypic spectrum is still
unclear, and due to the limited number of cases
reported so far, the existence ofRAX involvement
in syndromic forms of anophthalmia cannot be
excluded.
Acknowledgements
The authors are grateful to Dr Iba Zizen for providing magnetic
resonance imaging scan pictures.
References
1. Morrison D, FitzPatrick D, Hanson I et al. National study
of microphthalmia, anophthalmia, and coloboma (MAC)
in Scotland: investigation of genetic aetiology. J Med Genet
2002: 39: 16–22.
2. Lowry RB, Kohut R, Sibbald B et al. Anophthalmia and
microphthalmia in the Alberta Congenital Anomalies
Surveillance System. Can J Ophthalmol 2005: 40: 38–44.
3. Fantes J, Ragge NK, Lynch SA et al. Mutations in SOX2
cause anophthalmia. Nat Genet 2003: 33: 461–463.
4. RaggeNK, Lorenz B, Schneider A et al. SOX2 anophthalmia
syndrome. Am J Med Genet A 2005: 135: 1–7; discussion 8.
5. Bakrania P, Efthymiou M, Klein JC et al. Mutations in
BMP4 cause eye, brain, and digit developmental anomalies:
overlap between the BMP4 and hedgehog signaling path-
ways. Am J Hum Genet 2008: 82: 304–319.
6. Verma AS, Fitzpatrick DR. Anophthalmia and micro-
phthalmia. Orphanet J Rare Dis 2007: 2: 47.
7. Furukawa T, Kozak CA, Cepko CL. rax, a novel paired-
type homeobox gene, shows expression in the anterior
neural fold and developing retina. Proc Natl Acad Sci U S A
1997: 94: 3088–3093.
8. Zhang L, Mathers PH, Jamrich M. Function of Rx, but not
Pax6, is essential for the formation of retinal progenitor
cells in mice. Genesis 2000: 28: 135–142.
9. Mathers PH, Grinberg A, Mahon KA et al. The Rx
homeobox gene is essential for vertebrate eye development.
Nature 1997: 387: 603–607.
10. Bailey TJ, El-Hodiri H, Zhang L et al. Regulation of
vertebrate eye development by Rx genes. Int J Dev Biol
2004: 48: 761–770.
11. Voronina VA, Kozhemyakina EA, O’Kernick CM et al.
Mutations in the human RAX homeobox gene in a patient
with anophthalmia and sclerocornea. Hum Mol Genet
2004: 13: 315–322.
12. Tucker P, Laemle L, Munson A et al. The eyeless mouse
mutation (ey1) removes an alternative start codon from the
Rx/rax homeobox gene. Genesis 2001: 31: 43–53.
13. Loosli F, Staub W, Finger-Baier KC et al. Loss of eyes in
zebrafish caused by mutation of chokh/rx3. EMBO Rep
2003: 4: 894–899.
14. Andreazzoli M, Gestri G, Angeloni D et al. Role of Xrx1 in
Xenopus eye and anterior brain development. Develop-
ment 1999: 126: 2451–2460.
15. Harries LW, Bingham C, Bellanne-Chantelot C et al. The
position of premature termination codons in the hepatocyte
nuclear factor-1 beta gene determines susceptibility to
nonsense-mediated decay. Hum Genet 2005: 118: 214–224.
16. Amendt BA, Sutherland LB, Russo AF. Multifunctional
role of the Pitx2 homeodomain protein C-terminal tail. Mol
Cell Biol 1999: 19: 7001–7010.
17. Norris RA, Kern MJ. Identification of domains mediating
transcription activation, repression, and inhibition in the
paired-related homeobox protein, Prx2 (S8). DNA Cell
Biol 2001: 20: 89–99.
18. Eggert T, Hauck B, Hildebrandt N et al. Isolation of
a Drosophila homolog of the vertebrate homeobox gene Rx
and its possible role in brain and eye development. Proc
Natl Acad Sci U S A 1998: 95: 2343–2348.
19. Andreazzoli M, Gestri G, Cremisi F et al. Xrx1 controls
proliferation and neurogenesis in Xenopus anterior neural
plate. Development 2003: 130: 5143–5154.
20. Spieler D, Baumer N, Stebler J et al. Involvement of Pax6
and Otx2 in the forebrain-specific regulation of the
vertebrate homeobox gene ANF/Hesx1. Dev Biol 2004:
269: 567–579.
21. Martinez-Barbera JP, Rodriguez TA, Beddington RS. The
homeobox gene Hesx1 is required in the anterior neural
ectoderm for normal forebrain formation. Dev Biol 2000:
223: 422–430.
22. Dattani MT, Martinez-Barbera JP, Thomas PQ et al.
Mutations in the homeobox gene HESX1/Hesx1 associated
with septo-optic dysplasia in human and mouse. Nat Genet
1998: 19: 125–133.
RAX mutations in anophthalmia
395