Download Novel FGF8 Mutations Associated with Recessive

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G e n e t i c s — E n d o c r i n e
O N L I N E
R e s e a r c h
Novel FGF8 Mutations Associated with Recessive
Holoprosencephaly, Craniofacial Defects, and
Hypothalamo-Pituitary Dysfunction
Mark J. McCabe,* Carles Gaston-Massuet,* Vaitsa Tziaferi, Louise C. Gregory,
Kyriaki S. Alatzoglou, Massimo Signore, Eduardo Puelles, Dianne Gerrelli,
I. Sadaf Farooqi, Jamal Raza, Joanna Walker, Scott I. Kavanaugh, Pei-San Tsai,
Nelly Pitteloud, Juan-Pedro Martinez-Barbera, and Mehul T. Dattani
Developmental Endocrinology Research Group (M.J.M., V.T., L.C.G., K.S.A., M.T.D.), Clinical and
Molecular Genetics Unit, University College London—Institute of Child Health, and Neural Development
Unit (C.G.-M., M.S., D.G., J.-P.M.-B.), University College London—Institute of Child Health, London
WC1N 1EH, United Kingdom; Institute of Neurosciences (E.P.), Consejo Superior de Investigaciones
Científicas and Universidad Miguel Hernandez, 03202 Alicante, Spain; University of Cambridge Medical
Research Laboratories (I.S.F.), Institute of Metabolic Science, Addenbrooke’s Hospital, Cambridge CB2
OQQ, United Kingdom; National Institute of Child Health (J.R.), Karachi 75510, Pakistan; Department of
Paediatrics (J.W.), St. Mary’s Hospital, Portsmouth PO6 3LY, United Kingdom; Department of Integrative
Physiology (S.I.K., P.-S.T.), University of Colorado, Boulder, Colorado 80309-0354; and Department of
Endocrinology, Diabetes, and Metabolism (N.P.), University of Lausanne, 1005 Lausanne, Switzerland
Context: Fibroblast growth factor (FGF) 8 is important for GnRH neuronal development with
human mutations resulting in Kallmann syndrome. Murine data suggest a role for Fgf8 in hypothalamo-pituitary development; however, its role in the etiology of wider hypothalamo-pituitary
dysfunction in humans is unknown.
Objective: The objective of this study was to screen for FGF8 mutations in patients with septo-optic
dysplasia (n ⫽ 374) or holoprosencephaly (HPE)/midline clefts (n ⫽ 47).
Methods: FGF8 was analyzed by PCR and direct sequencing. Ethnically matched controls were then
screened for mutated alleles (n ⫽ 480 – 686). Localization of Fgf8/FGF8 expression was analyzed by
in situ hybridization in developing murine and human embryos. Finally, Fgf8 hypomorphic mice
(Fgf8loxPNeo/⫺) were analyzed for the presence of forebrain and hypothalamo-pituitary defects.
Results: A homozygous p.R189H mutation was identified in a female patient of consanguineous
parentage with semilobar HPE, diabetes insipidus, and TSH and ACTH insufficiency. Second, a
heterozygous p.Q216E mutation was identified in a female patient with an absent corpus callosum,
hypoplastic optic nerves, and Moebius syndrome. FGF8 was expressed in the ventral diencephalon
and anterior commissural plate but not in Rathke’s pouch, strongly suggesting early onset hypothalamic and corpus callosal defects in these patients. This was consolidated by significantly reduced vasopressin and oxytocin staining neurons in the hypothalamus of Fgf8 hypomorphic mice
compared with controls along with variable hypothalamo-pituitary defects and HPE.
Conclusion: We implicate FGF8 in the etiology of recessive HPE and potentially septo-optic dysplasia/
Moebius syndrome for the first time to our knowledge. Furthermore, FGF8 is important for the development of the ventral diencephalon, hypothalamus, and pituitary. (J Clin Endocrinol Metab 96:
E1709 –E1718, 2011)
ISSN Print 0021-972X ISSN Online 1945-7197
Printed in U.S.A.
Copyright © 2011 by The Endocrine Society
doi: 10.1210/jc.2011-0454 Received February 18, 2011. Accepted July 12, 2011.
First Published Online August 11, 2011
* M.J.M. and C.G.-M. contributed equally to this work.
Abbreviations: AVP, Arginine vasopressin; CS, Carnegie stage; FGF, fibroblast growth
factor; FT4, free T4; HPE, holoprosencephaly; MRI, magnetic resonance imaging; OT, oxytocin; PN, postnatal day; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus; SDS,
SD score; SOD, septo-optic dysplasia; SON, supraoptic nucleus.
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FGF8 in HPE and Midline/Craniofacial Defects
omplex midline defects of the forebrain in humans are
rare but may be associated with hypopituitarism,
which in turn may lead to significant morbidity and mortality. They span a wide spectrum of phenotypes ranging
from those which are incompatible with life, to holoprosencephaly (HPE) and cleft palate and septo-optic dysplasia (SOD).
SOD is a highly heterogeneous condition which, although usually sporadic and inclusive of possible environmental (including drug and alcohol induced) pathologies,
has also been identified in a number of familial cases involving mutations in an increasing number of early developmental transcription factors including HESX1, SOX2,
SOX3, and OTX2 (1–5). These genes are expressed in
regions that determine the formation of forebrain and related midline structures such as the hypothalamus and
pituitary (6). Consequently, SOD is characterized by variable phenotypes including midline telencephalic abnormalities, optic nerve hypoplasia, and pituitary hypoplasia
with variable pituitary hormone deficiencies (7, 8).
HPE is etiologically heterogeneous but is the most frequent developmental forebrain anomaly in humans, with
an incidence in liveborns of approximately one in 10,000 –
20,000 and in conceptuses as high as one in 250 (9). It
results from varying degrees of incomplete cleavage of the
prosencephalon into the cerebral hemispheres and ventricles. In addition, failure of the frontal and parietal lobes to
divide posteriorly results in an absent corpus callosum.
Facial features associated with HPE include cyclopia,
anophthalmia, midface hypoplasia, hypotelorism, cleft lip
and/or palate, and a single central incisor (10).
Recent studies have implicated a number of heterozygous genetic missense mutations and deletions in the etiology of HPE; cytogenetically visible abnormalities are
estimated to be present in 25% of HPE patients (11). These
in turn have led to the identification of a number of causative genes, including SHH, ZIC2, SIX3, and TGIF1 with
subsequent identification of mutated genes in associated
pathways including PTCH1, GLI2, DISP1, TDGF1,
GAS1, EYA4, and FOXH1 (12–14). However, mutations
have been identified in only 17% of cytogenetically normal children with HPE. In recent studies, submicroscopic
deletions of a number of loci believed to be implicated in
HPE were identified in a number of individuals with HPE
(15), suggesting that a number of genetic mutations remain to be described.
Although not previously related to hypopituitarism,
Kallmann syndrome is classically defined as the association of hypogonadotrophic hypogonadism with anosmia
due to hypoplasia of the olfactory bulbs (16). However,
the condition is genetically and clinically heterogeneous
and may be associated with craniofacial defects such as
C
J Clin Endocrinol Metab, October 2011, 96(10):E1709 –E1718
Moebius syndrome, which is characterized by malformation of the sixth and seventh facial nerves (17, 18). One of
the genetic pathways involved in Kallmann syndrome is
the ubiquitously expressed fibroblast growth factor (FGF)
family of signaling molecules and its receptors (19). Lossof-function mutations in human FGF8 and FGFR1 have
been implicated in this condition, and these factors potentially link the disorder to hypopituitarism through the
requirement of Fgf8 to maintain anterior pituitary cellular
proliferation via Lhx3 in mice (20, 21). Recently a putative role for FGF8 in two patients with HPE has been
postulated upon the identification of two heterozygous
mutations: 1) a 138-kb deletion at 10q24.3 encompassing
FGF8 as well as a number of other genes (15), and 2) a
p.T229M substitution associated with incomplete penetrance, which has previously been described in association with isolated hypogonadotrophic hypogonadism (16,
22). Although the latter patient manifested diabetes insipidus, there was no other evidence of endocrine dysfunction
in the two pedigrees.
We hypothesized that FGF8 may be essential for the
development of the forebrain and pituitary gland in humans and mouse and that mutations in this gene may contribute to the etiology of disorders such as SOD and HPE.
Patients and Methods
Patients
A total of 421 patients with congenital hypopituitarism and
midline craniofacial/forebrain defects (SOD or HPE) were recruited between 1998 and 2010; 167 patients (39.7%) were recruited at the London Centre for Pediatric Endocrinology based
at Great Ormond Street Hospital for Children and the University
College London Hospital, and 157 (37.3%) were referred from
national and 97 (23%) from international centers. Ethical committee approval was obtained from the University College London Institute of Child Health/Great Ormond Street Hospital for
Children Joint Research Ethics Committee, and informed written consent was obtained from patients and/or parents. Of the
421 patients screened (male to female ratio 1.1:1), 88.8% (n ⫽
374) had SOD and its variants, whereas 11.2% (n ⫽ 47) had HPE
or midline clefts.
Mutation analysis
The coding region of human FGF8 (NM_033163) was amplified by PCR on an Eppendorf Thermocycler (Netheler, Germany) over 35 cycles with primers designed using the Primer3
program (available at http://frodo.wi.mit.edu/primer3) flanking
each of the coding regions, including the four alternative splice
variants of exon 1 (Fig. 1). PCR parameters are available on
request. For direct sequencing for mutations, PCR products were
treated with MicroClean reagent (Web Scientific, Cheshire, UK;
catalog no. 2MCL-10) according to manufacturer’s instructions
and then sequenced using BigDye version 1.1 sequencing chemistry (Applied Biosystems, Warrington, UK) and analyzed on a
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Immunohistochemistry and
quantification of hypothalamic arginine
vasopressin (AVP)-expressing nuclei
FIG. 1. Structure of FGF8 isoforms. Schematic representation of the four FGF8
isoforms (A, B, E, and F) resulting from alternative splicing of exons 1C and 1D. The
lettering refers to the specific FGF8 isoform. Exons are designated inside the
rectangles and numbers above each exon-exon boundary indicate the amino acid
position in the FGF8f isoform. The core region of the protein is predominantly made
up of exons 2 and 3, which is conserved between isoforms. Previously identified
mutations are indicated by arrows and numbered according to the FGF8f protein
isoform, with the two novel mutations presented within this paper in blue type. Note
that the asterisk denotes homozygous mutations. [Adapted from J. Falardeau et al.:
Decreased FGF8 signaling causes deficiency of gonadotropin-releasing hormone in
humans and mice. J Clin Invest 118:2822–2831, 2008 (16), with permission.
© American Society for clinical Investigation; and E. Trarbach et al.: Nonsense
mutations in FGF8 gene causing different degrees of human gonadotropin-releasing
deficiency. J Clin Endocrinol Metab 95:3491–3496, 2010 (23), with permission.
© The Endocrine Society.
3730X1 DNA analyzer (Applied Biosystems/Hitachi, Tokyo, Japan; catalog no. 625-0020). Upon discovering FGF8 mutations,
480 – 686 controls were screened at the same allele to provide
comparisons.
In situ hybridization and hypomorphic mice
In situ hybridization was performed on paraffin sections from
mouse and human embryos as previously described (24). Sections of human embryos were obtained from the Human Developmental Biology Resource (University College London Institute
of Child Health, London, UK). The human FGF8 and FGFR1
probes were obtained from the Source BioScience LifeSciences
(http://www.lifesci.ences.sourcebioscience.com/). The Fgf8 hypomorphic mouse embryos (Fgf8loxPNeo/⫺) were obtained by
crossing the Fgf8⫹/⫺ with Fgf8loxPNeo/⫹ line as has previously
been described (25).
Brains from wild-type, heterozygote, and homozygote Fgf8 hypomorphic mouse embryos
(Fgf8loxPNeo/⫺) were collected at postnatal day (PN)
0 and processed for immunohistochemistry on
50-␮m frozen sections. Sections were taken from the
preoptic area to the mamillary bodies, and neurons
were stained for AVP using a rabbit anti-AVP antibody (Millipore, Billerica, CA; catalog no. AB1565)
for 48 h at 4 C. These were then sequentially incubated with a biotinylated donkey antirabbit secondary antibody (1:500; Jackson ImmunoResearch,
West Grove, PA) and the avidin-biotin complex
(Vector Laboratories, Burlingame, CA). Immunoreactivity was visualized with diaminobenzidine as
the chromagen before counterstaining with methyl
green.
Quantification of AVP-immunoreactive paraventricular nuclei (PVN) and suprachiasmatic
nuclei (SCN) were conducted as recently described (26).
Statistical analysis
Treatments presented (see Fig. 4T) were compared with controls by ANOVA followed by Student Newman-Keuls test. P ⬍ 0.05 was used to
determine whether results were statistically significant. All statistics were performed using SigmaStat version 3.5 (Systat Software, Inc., San
Jose, CA).
Results
Mutation analysis
After direct sequencing of 421 patients, two unrelated female patients were detected with novel mutations in exon 3 of FGF8. These included: 1) a homozygous mutation, c.566G⬎A, resulting in the substitution
of arginine by histidine (p.R189H) (patient 1) and 2) a
heterozygous mutation, c.646C⬎G, resulting in the
substitution of glutamine by glutamic acid (p.Q216E)
(patient 2). The clinical characteristics of both patients
TABLE 1. Clinical characteristics of affected patients with FGF8 mutations
Patient
no.
Mutation
1
Homozygous
2
Position
Sex Hypothalamo-pituitary
MRI
Other
c.566G⬎A, p.R189H F DI, ACTHI, evolving TSHD, Semilobar HPE,
DD, high arched palate,
PRLD
bulky AP
maxillary hypoplasia
Heterozygous c.646C⬎G, p.Q216E F Borderline GH response
Agenesis of CC, optic Moebius syndrome,
to provocation
nerve hypoplasia,
microcephaly, DD,
normal pituitary
spastic diplegia
DI, Diabetes insipidus; ACTHI, ACTH insufficiency; TSHD, TSH deficiency; PRLD, prolactin deficiency; CC, corpus callosum; DD, developmental
delay; AP, anterior pituitary; F, female.
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FIG. 2. Two novel mutations associated with recessive HPE and SOD. A and B, A total of 421 patients with hypopituitarism phenotypes ranging
from HPE to SOD were screened for mutations in FGF8 by direct sequencing. A female patient with HPE exhibited a novel homozygous mutation
at position c.566, resulting in a p.R189H substitution (A, arrow) in a highly conserved residue in all vertebrates analyzed (B). C and D, A second
female patient with SOD and Moebius syndrome exhibited a novel heterozygous mutation at position c.646, leading to a p.Q216E substitution (C,
arrow) in a highly conserved amino acid among mammals (D). E, Sagittal MRI scan of our SOD/Moebius syndrome patient (Q216E), demonstrating
an absent corpus callosum (asterisk) but otherwise normal anterior pituitary (AP) and posterior pituitary (PP) glands. F, Sagittal and coronal MRI
scans of a normal control subject and our HPE (p.R189H) patient. The latter presented with an absent corpus callosum (asterisk) and an enlarged
anterior pituitary (AP). Failure of the brain to divide into its cerebral hemispheres in the patient is apparent in the coronal sections with a clearer
view of the absent corpus callosum in the far right image.
are presented in Table 1. None of 480 Caucasian controls screened exhibited either of the mutations. In addition, for patient 1, who was of Pakistani origin,
screening a further 206 ethnically matched controls revealed no mutations. Neither patient identified with an
FGF8 mutation had mutations in FGFR1, KAL1,
PROK2, PROKR2, HESX1, or SHH.
Patient 1
A female patient from a consanguineous family of Pakistani origin had been diagnosed antenatally with holoprosencephaly and absent corpus callosum; at birth she
was noted to have microcephaly, micrognathia, and a high
arched palate. She presented at the age of 7 wk with seizures and hypernatremia and was admitted to intensive
care. Although her critical condition did not allow for
extensive endocrine investigations, she was diagnosed
with diabetes insipidus and ACTH insufficiency and has
been treated with desmopressin and hydrocortisone. A
brain magnetic resonance imaging (MRI) confirmed semilobar holoprosencephaly with separation of the cerebral
hemispheres anteriorly and failure of separation of the
basal ganglia structures and the thalami from each other
and across the midline. The corpus callosum was absent,
and the hypothalamus was abnormal. The anterior pituitary gland was noted to be bulky, and the posterior pituitary was identified at the normal location (Fig. 2F).
A glucagon stimulation test performed at the age of 5.5
yr [height 107.6 cm (⫺0.6 SD score [SDS]), weight 16.6 kg
(⫺0.9SDS)] showed a normal GH response (peak GH 29.1
␮g/liter) with normal IGF-I (148 ng/ml; normal range 52–
297 ng/ml) and IGF binding protein-3 (3.19 mg/liter; normal range 1.3–5.6 mg/liter), but she gradually developed
TSH deficiency as shown by persistently low free T4 (FT4)
concentrations, with a progressive reduction in prolactin
concentrations (Table 2).
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TABLE 2. Serial endocrine evaluation of patient 1 (p.R189H)
Age
8 wk
21 months
4 yr
5.5 yr
6.5 yr
7 yr
Cortisol
(␮g/dl)
⬍1–5.3 on profile
ND
ND
18 (peak GST
on hydrocortisone)
ND
ND
Peak GH
TSH
(␮g/liter)
(mU/liter)
(NR <6mU/ Basal PRL PRL NR Basal LH Basal FSH
(NR >6.7
FT4
liter)
(␮g/liter) (␮g/liter) (IU/liter) (IU/liter)
␮g/liter) (ng/dl) FT4 NR
ND
1.63 1.09 –1.78
7.4
70.5
4.9 – 67
⬍0.7
2.7 IU/liter
ND
1.24 1.09 –1.78
3.6
ND
ND
ND
ND
0.96
0.93–1.71
1.4
6.79
3.1–11.1
ND
ND
29.1
1.00 0.84 –1.47
1.1
3.34
3.1–11.1
⬍0.2
0.8
ND
ND
0.98
0.79
1.00 –1.65
0.84 –1.47
0.88
1.2
ND
ND
ND
GST, Glucagon stimulation test; PRL, prolactin; ND, not done; NR, normal range.
Genetic screening revealed that she was homozygous
for a novel FGF8 mutation (c.566G⬎A, p.R189H) involving a highly conserved region of the gene (Fig. 2, A and
B). Both parents were heterozygous and had a normal
phenotype with no history of delayed puberty or subfertility and a reported normal sense of smell.
Patient 2
A Caucasian female patient with SOD and agenesis of
corpus callosum presented at the age of 6 yr for investigation of profound growth deceleration with a height of
99.2 cm (⫺2.7 SDS) and a weight of 15.8 kg (⫺1.9 SDS).
She was born to unrelated parents, and in addition to
SOD, she had microcephaly, global developmental delay
with evolving spastic diplegia, and Moebius syndrome.
Endocrine investigations revealed a low normal concentration of IGF-I [107 ␮g/liter (normal range: 53–302␮g/
liter)] with borderline peak GH responses to glucagon and
clonidine stimulation (5.13 and 5 ␮g/liter, respectively),
normal thyroid function, and an adequate peak cortisol in
response to both glucagon (16.1 ␮g/dl) and synacthen (30
␮g/dl) stimulation. Brain MRI revealed an absent corpus
callosum and hypoplastic optic nerves with no structural
abnormality of the pituitary gland (Fig. 2E). She had no
evidence of any midline cleft, nor did she manifest diabetes
insipidus. The patient is currently too young to test for
olfactory dysfunction.
The patient was heterozygous for a novel FGF8 mutation (c.646C⬎G, p.Q216E) (Fig. 2C) in a highly conserved
region of the gene across multiple species (Fig. 2D),
whereas her unaffected mother was a carrier.
Expression analysis of FGF8 and FGFR1 in the
developing human embryo
To understand the role of FGF8 and FGFR1 in human
embryonic development and to bring insights into the
pathogenesis of the phenotypes observed in patients harboring mutations in these genes, we performed expression
analysis of FGF8 and FGFR1 in human embryos.
In situ hybridization analyses on early-staged human
embryos, Carnegie stage (CS) 16, revealed FGF8 mRNA
transcripts in the midline commissural plate of the developing telencephalon and telencephalic/diencephalic border and in the ventral diencephalon, but no expression was
observed in Rathke’s pouch (the primordium of the anterior pituitary) (Fig. 3A). In mouse, expression of Fgf8 in
the ventral diencephalon is essential for Rathke’s pouch
induction and growth (21, 27, 28). In agreement with this
notion, expression of FGFR1, which is the main receptor
mediating FGF8 signaling, was detected in Rathke’s
pouch (Fig. 3, B and D) and in a broader domain within the
ventral diencephalon (Fig. 3B). At CS 22, FGFR1 transcripts were present in the neuroepithelium of the developing brain and in Rathke’s pouch (Fig. 3E). FGF8 and
FGFR1 expression patterns in the human embryo parallel
that of the mouse, suggesting a conserved function for
FGF8 and FGFR1 in the development of these structures.
Hypothalamic and pituitary defects in mouse
embryos carrying a hypomorphic Fgf8 allele
Mouse embryos carrying a hypomorphic allele of Fgf8
(Fgf8-loxPNeo) over a null allele (Fgf8loxPNeo/⫺) show severe defects in brain morphogenesis, including midbrain
defects, absence of olfactory bulbs and optic chiasm, and
HPE with an abnormal corpus callosum [Meyers et al.
(25); Storm et al. (29)]. To further understand the pathogenesis of the defects observed in the patients carrying
FGF8 mutations, we analyzed by in situ hybridization the
differentiation of hormone-producing cells in the pituitary
gland and the integrity of the neuroendocrine hypothalamus in Fgf8 hypomorphic mouse embryos. In addition to
HPE, which was observed in two of six mutants analyzed,
Fgf8loxPNeo/⫺ embryos showed severe abnormalities in the
pituitary gland and endocrine hypothalamus (Fig. 4). At
17.5 d postcoitum, we observed two different phenotypes:
1) a severe one, characterized by the substantial reduction
of anterior pituitary tissue and absence of posterior lobe
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J Clin Endocrinol Metab, October 2011, 96(10):E1709 –E1718
region of the protein, in a patient with
semilobar HPE associated with an absent corpus callosum and a bulky anterior pituitary on MRI. The parents of
our proband are both unaffected carriers of the mutation and are first cousins.
The recessive nature of this mutation,
which occurs at a highly conserved residue, and its absence in 686 controls,
suggests that FGF8 is critical for normal
forebrain development, as indicated in
a murine model carrying a hypomorphic Fgf8 allele (25, 29, 30).
The affected region of the protein is conserved across all isoforms and the variably
FIG. 3. Expression of FGF8 and FGFR1 during early human development. Expression of FGF8 (A)
penetrant loss-of-function heterozygous
and FGFR1 (B–E) during human embryonic CS 16 [37 d after fertilization (A and B) and CS 22 (C,
D, and E)]. Sagittal (A–D) and coronal sections (E) are shown. A, FGF8 transcripts are localized in
T229M mutation, which lies in the same
the commissural plate of the telencephalon (arrow in A), telencephalic/diencephalic border
region, has previously been associated
(asterisk), and the prospective hypothalamus (arrowheads in A) but not in Rathke’s pouch (rp, blue
with hypogonadotrophic hypogonadism
arrow), the primordium of the anterior pituitary gland. B–E, FGFR1 transcripts are widely detected
and HPE (16, 22). However, in vitro lithroughout the brain neuroepithelium (C and E) including the prospective hypothalamus (arrows
in B, D, and E), hindbrain (arrowheads in B), and the developing Rathke’s pouch (blue arrow in B,
gand receptor binding assays using bacrp in D and E). D, An enlarged image of the boxed area in C. Scale bar (B), 200 ␮m; (C), 2 mm.
teriological recombinant proteins appear
to indicate that this FGF8 region may
(two of six embryos analyzed) (Fig. 4, B, E, H, K, and N); not be important for receptor binding (30). This sugand 2) a mild phenotype, in which the pituitary gland was gests that FGF8 activity in vivo may require posttransmorphologically more comparable with the controls (four lational modifications, which were absent in the recomof six embryos analyzed) (Fig. 4, C, F, I, L, and O). Despite binant proteins used in the previous study, but
these morphological abnormalities, the only defects in ter- important for other aspects of FGF8 biology such as
minal differentiation of the hormone-producing cells were intracellular transport, diffusion, or protein half-life.
observed in those secreting LH.
Mouse studies strongly suggest that fine-tuning control
Next, we studied the integrity of the endocrine hypo- of FGF8 signaling through gene dosage and binding to
thalamus in the Fgf8 hypomorph embryos. A marked re- secreted inhibitors is essential for normal development.
duction of AVP and oxytocin (OT) neurons was observed
The assessment of the impact of the p.R189H mutation
in the supraoptic (SON), SCN, and PVN nuclei in these
identified in this study on normal development awaits
mutant embryos relative to the controls (Fig. 4, P–S).
the development of new mouse models in the future.
Quantification of AVP-immunoreactive neurons on PN0
Recently a role for FGF8 in the etiology of HPE has been
confirmed a significant (P ⬍ 0.002) loss of these neurons
proposed (15). However, because a cause-effect relationin the SCN and PVN of FGF8 hypomorphs (Fig. 4T).
ship was not established and mutations were found in
Together these expression analyses in mouse embryos
heterozygosity, the possibility exists that mutations in
highlight an essential role for FGF8 signaling in the forother genes may underlie the observed defects (digenicity).
mation of the hypothalamic-pituitary axis and provide
Although loss-of-function homozygous mutations in
insights into the pathogenesis of the brain and endocrine
FGF8 have been identified or predicted previously in padefects observed in patients carrying mutations in FGF8
tients with normosmic idiopathic hypogonadotrophic hyand FGFR1.
pogonadism (16) and nonsyndromic cleft lip and palate,
respectively (31), our patient is the first with a recessive
form of HPE to be associated with an FGF8 mutation; all
Discussion
the other genetic mutations implicated in the disorder have
been transmitted in a heterozygous state with variable
Novel homozygous mutation in FGF8 in a female
penetrance. The lack of a phenotype in the heterozygous
with holoprosencephaly
We have identified a novel homozygous mutation, parents is particularly intriguing, given that heterozygous
c.566G⬎A (p.R189H), in exon 3 of FGF8, which resulted mutations in the gene have previously been associated
in an arginine substitution for histidine in the C-terminal with Kallmann syndrome.
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FIG. 4. Mouse embryos carrying a Fgf8 hypomorphic allele exhibit pituitary and hypothalamic defects. In situ hybridization with specific markers
on pituitary (A–O) or hypothalamic (P–S) frontal histological sections. A–O, Terminal differentiation of somatotrophs [Gh expressing cells (A–C)],
corticotrophs and melanotrophs [Pomc1 expressing cells, D–F)], and thyrotropes and gonadotrophs [Cga expressing cells (G–I)] is unaffected in the
Fgf8 hypomorph (Fgf8loxPNeo/⫺) embryos. However, the pituitary gland is smaller in the Fgf8 hypomorph embryos relative to Fgf8⫹/⫹ controls. Note
the absence of posterior lobe in one embryo (arrow in B, E, and H). Expression of Tsh␤ was unaffected across the genotypes (J–L). However, a
proportion of Fgf8 hypomorphs exhibited lack of Lh␤-expressing cells (M–O). P–S, The endocrine hypothalamus is severely compromised in Fgf8
hypomorph embryos as revealed by in situ hybridization with AVP (P, P⬘, Q, and Q⬘) and OT (R, R⬘, S, and S⬘) antisense riboprobes. There is an
apparent reduction in numbers of AVP- and OT-expressing neurons in the SON, SCN, and PVN in the Fgf8 hymomorph embryos compared
with control littermates. P⬘, Q⬘, R⬘, and S⬘ are magnified images of regions boxed in P, Q, R, and S of the same or an alternative section. T,
Quantification analysis of AVP-immunoreactive neurons demonstrates a significant reduction in numbers of these neurons in the SCN and PVN
nuclei of PN0 mice carrying specific combinations of Fgf8 alleles. Wt is Fgf8⫹/⫹, Het is Fgf8loxpNeo/⫹, and Homo is Fgf8loxPNeo/⫺. AL, Anterior lobe;
PL, posterior lobe;. Scale bars, 200 ␮m. Data are mean ⫾ SD. **, P ⬍ 0.002.
Novel heterozygous mutation in FGF8 in a female
with SOD and Moebius syndrome
The patient with the heterozygous p.Q216E substitution, which lies in the same region as our homozygous
R189H mutation, manifested midline defects including an
absent corpus callosum and hypoplastic optic nerves, reflecting a variant of SOD. A borderline GH response to
stimulation with a low-normal IGF-I was the only endocrine abnormality, although one cannot exclude the possibility of an evolving endocrinopathy, particularly with
reference to gonadotropin secretion.
An unusual finding in this patient was the presence of
Moebius syndrome, a rare congenital dysinnervation syndrome characterized by poorly developed abducens (VI)
E1716
McCabe et al.
FGF8 in HPE and Midline/Craniofacial Defects
and facial (VII) nerves (17). The condition has been associated with Kallmann syndrome in five cases (18), with the
only midline association reported recently in a child with
holoprosencephaly in which discontinuation of the pregnancy had been attempted with misoprostol, an agent previously linked with Moebius syndrome (32, 33). Although
FGF8 has never previously been implicated in this disorder, its potential role is highlighted by its expression in the
facial primordia and eyes in mice and the presence of facial
skeletal defects in heterozygote Fgf8 zebrafish mutants,
which resemble several human craniofacial disorders (34,
35). Furthermore, tissue-specific ablation of Fgf8 was associated with abnormal neural crest survival and patterning, which gives rise to the cranial nerves and bone, cartilage, connective tissue, and skin pigmentation (36). In
agreement with this finding, the Fgf8loxPNeo/⫺ embryos
analyzed in this study also presented craniofacial defects.
The unaffected mother of the patient was the heterozygous carrier of the FGF8 mutation, suggesting variable
penetrance of the mutated allele, or possibly a digenic
cause of the disorder, both of which have been associated
with Kallmann syndrome due to FGF8 mutations. Cases
of the former are not unusual, with variable phenotypicpresentations within a given family, making it difficult to
predict an expected phenotype based on the genotype
alone (37). Variations in environmental exposure may
also contribute to phenotypic variations (38). Alternatively, more than one gene could contribute to the condition, particularly in the most severe form (39). Screening
for mutations in HESX1 and SHH, as well as other Kallmann syndrome-associated genes, failed to establish a digenic cause, although there may be a contribution from an
as-yet-unknown gene, given that no genetic etiology has
been identified in the majority of patients with Kallmann
syndrome or midline forebrain defects. The association of
Moebius syndrome with SOD in our patient, and with
Kallmann syndrome and HPE in other patients, implicates
an intrinsic link between these disorders in the form of
overlapping phenotypes; both our data and those published previously point to associations with mutated FGF8
or related genes.
Fgf8/FGF8 is expressed in the developing
hypothalamus and implicated in normal
hypothalamo-pituitary development
Our expression analysis demonstrates that Fgf8/FGF8
is expressed in the telencephalon and ventral diencephalon
(prospective hypothalamus) but not in Rathke’s pouch,
which is destined to form the anterior pituitary (39). Expression of FGFR1 colocalizes with FGF8 in the developing hypothalamus and is also found in Rathke’s pouch.
The similar localization of Fgf8/FGF8 expression in both
J Clin Endocrinol Metab, October 2011, 96(10):E1709 –E1718
mice and humans as presented herein suggests that the
disrupted pituitary and hypothalamus in the Fgf8 hypomorphic mouse may parallel the endocrine deficits observed in our patients. The patient with the homozygous
FGF8 mutation presented a bulky anterior pituitary with
the presence of a posterior pituitary despite the association
with diabetes insipidus, on MRI, and of note, the murine
hypomorphs showed marked variability in the size of the
anterior pituitary and the presence or otherwise of the
posterior pituitary. Additionally, the reduction in AVP in
the magnocellular SON and PVN of the hypomorphs may
correlate with the diabetes insipidus observed in our patient, which is due to insufficient vasopressin signaling, a
hormone important for maintaining urinary salt/water reabsorption (40). Furthermore, Fgf8 hypomorphs also harbor defects in the parvicellular AVP neurons in the SCN,
suggesting that other hypothalamic functions such as circadian output may be dysregulated. Although not quantified in this study, the reduction in OT staining in SON
and PVN nuclei is consistent with recently published data,
which showed significantly reduced OT immunoreactivity
in the same hypomorphic mice as used herein (26). Our
data suggest that the hypopituitary phenotypes observed
in our patients may be the result of reduced functional
FGF8 in the diencephalon, leading to deficiencies in the
neuroendocrine hypothalamus.
In conclusion, we report mutations in FGF8 that are
associated with complex midline phenotypes, including
the first autosomal recessive case of HPE and a potential
role in a patient with SOD and Moebius syndrome, the
genetic basis of the latter having remained elusive to date.
Analysis of our patients and murine hypomorphs, as well
as the conserved expression patterns of Fgf8/FGF8 and
Fgfr1/FGFR1 in mouse and human, suggests that FGF8
signaling is important for telencephalic and hypothalamopituitary axis development and that mutations in this gene
play a role in the pathogenesis of midline defects such as
HPE and SOD as well as Kallmann syndrome.
Acknowledgments
Human embryonic material was provided by the Human Developmental Biology Resource (www.hdbr.org), which is supported by the Medical Research Council Grant G0700089 and
the Wellcome Trust Grant 082557. We thank Dr. Gail Martin
and Dr. Mark Lewandosky for providing mouse embryos.
Address all correspondence and requests for reprints to: Mehul Dattani, Head of Endocrinology, Institute of Child Health
and Great Ormond St. Children’s Hospital, Pediatric Endocrinology, 30 Guilford Street, London WC1N 1EH, United Kingdom. E-mail: [email protected].
J Clin Endocrinol Metab, October 2011, 96(10):E1709 –E1718
This work was supported in part by a grant from the British
Society for Pediatric Endocrinology and Diabetes awarded to
V.T. and M.T.D. I.S.F. is supported by the Wellcome Trust,
Medical Research Council Centre for Obesity and Related Disorders and the U.K. National Institute for Health Research Cambridge Biomedical Research Centre. M.J.M. is supported by a
project grant from the Birth Defects Foundation-NewLife.
M.T.D. is also funded by Great Ormond Street Children’s Charity. J.-P.M.-B. is supported by Wellcome Trust Grants 084361
and 086545. E.P. is supported by El Ministerio de Ciencia e
Innovación Grant BFU2010-16538. C.G.-M. is supported by the
National Institute for Health Research Great Ormond Street
Hospital for Children/Univeristy College London Institute of
Child Health Specialist Biomedical Research Centre.
Disclosure Summary: The authors have nothing to disclose.
jcem.endojournals.org
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