American Journal of Medical Genetics 112:176–180 (2002)
Craniosynostosis in Alagille Syndrome
Binita M. Kamath,1Catherine Stolle,3Lynn Bason,2Raymond P. Colliton,2David A. Piccoli,1
Nancy B. Spinner,2and Ian D. Krantz2*
1Division of Gastroenterology and Nutrition, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania
2Division of Human Genetics and Molecular Biology, The Children’s Hospital of Philadelphia, Philadelphia,
3The University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Alagille syndrome is a multisystem develop-
mental disorder with primary involvement
of the liver, heart, skeleton, eyes and facial
structures, and demonstrates highly vari-
able expressivity with respect to all of the
involved systems. Alagille syndrome is cau-
sed by mutations in the Jagged1 gene.
Jagged1 is a ligand in the Notch signaling
pathway that has been shown to regulate
early cell fate determination. Mutations in
Jagged1 have been identified in approxi-
mately 80% of patients with Alagille syn-
drome. We have recently identified two
patients with mutation proven Alagille syn-
drome who also had unilateral coronal
screened for mutations in fibroblast growth
factor receptor 1, 2, 3 and TWIST genes, all
associated with various types of craniosy-
nostosis and no mutations were identified.
The finding of a conserved form of craniosy-
nostosis in two unrelated patients with
nial suture formation.
? 2002 Wiley-Liss, Inc.
KEY WORDS:Alagille syndrome; Jagged1;
Alagille syndrome (AGS, OMIM 118450) is a complex
dominantly inherited multisystem disorder involving
predominately the liver, heart, eyes, face and skeleton
[Krantz et al., 1999]. AGS demonstrates highly variable
expressivity with respect to all of the involved systems
gene, which maps to chromosome 20p12, have been
Notch signalling pathway that has been shown to
regulate early cell fate determination. We have identi-
fied two patients with AGS who also have unilateral
craniosynostosis. Abnormalities of the skeletal system
have been well documented in AGS [Rosenfield et al.,
1980]. Butterfly vertebrae are the most common bony
abnormality described and are seen in approximately
is the first report of craniosynostosis in individuals with
Craniosynostosis, the premature fusion of cranial
sutures, has been classified in many ways. In broad
terms it may be simple or complex. In simple craniosy-
nostosis, one suture is prematurely fused whereas
complex craniosynostosis describes the situation in
which two or more sutures are fused. Furthermore,
craniosynostosis may be either isolated or syndromic.
In isolated craniosynostosis the individual has no
other abnormalities other than those that occur secon-
darily to early sutural obliteration. Syndromic cranio-
synostosis describes an individual who also has other
primary defects of morphogenesis. The underlying
basis of many forms of syndromic craniosynostosis has
been defined on a molecular level. Mutations in the
fibroblast growth factor receptor 1, 2 and 3 (FGFR1, -2
and -3) and TWIST genes have been identified in
several syndromic forms of craniosynostosis [Cohen,
1995]. However there remain a significant number
of individuals with craniosynostosis in whom no
genetic cause can be found. This description of cranio-
synostosis in 2 unrelated individuals with mutation
proven AGS suggests a role for JAG1 in cranial suture
Grant sponsor: NIDDK; Grant numbers: 1K08 DK02541-01 (to
I.D.K.), NIDDK 1 R01 DK53104 (to N.B.S.); Grant sponsor: The
Fred and Saranne Biesecker Center For pediatric Liver Disease at
the Children’s Hospital of Philadelphia.
*Correspondence to: Ian D. Krantz, M.D., Division of Human
Genetics and Molecular Biology, 1002 Abramson Research
Building, The Children’s Hospital of Philadelphia, 34th Street
and Civic Center Blvd., Philadelphia, PA 19104.
Received 22 January 2002; Accepted 4 February 2002
? 2002 Wiley-Liss, Inc.
Patient A, a female, was born to a 32-year-old G3P3
Caucasian woman with no prior obstetric or medical
history of note. The parents were non-consanguinous
and the father was of Hispanic origin. The pregnancy
tension, which required hospitalization for observation,
but no medication. Patient A was born at 37 weeks
gestation by Cesarean section with no complications.
48 cm (25thcentile). A cardiac murmur was noted at
3 days and a subsequent echocardiogram revealed
tetralogy of Fallot. A modified right Blalock-Taussing
jaundiced with a total bilirubin of 8.1 mg/dL and a
conjugated fraction of 6.3 mg/dL. An ophthalmological
examination at this time was unremarkable. Radiologi-
cal examination of the spine revealed butterfly verteb-
rae. No confirmed diagnosis regarding the jaundice
Patient A remained persistently jaundiced. At the
age of 21 months a laparoscopic liver biopsy was
performed which revealed paucity of bile ducts and the
diagnosis of AGS was made. Patient A’s mother had
noticed an unusual skull shape in infancy, however
unilateral left coronal synostosis was not diagnosed
until 15 months age. This subsequently required
7 years. She had remained clinically well. The tetralogy
of Fallot was successfully corrected at the age of 2. Her
liver disease was stable and her main clinical complaint
was pruritis, particularly at night. On physical exam-
ination her height was 110 cm (<5thcentile). Her head
circumference was 46.5 cm (<3rdcentile; 50thcentile for
18 months). There was no frontal bossing or bitemporal
narrowing and her fontanelles were closed. She had
typical facial features of AGS, namely a prominent
forehead, deep-set eyes, straight nose with a flattened
flexion creases were also present.
A repeat opthalmological examination at this time
revealed bilateral posterior embryotoxon. In addition
she had an incomitant strabismus and bilateral tilted
which was consistent with the unilateral coronal
synostosis. The diagnosis of AGS was confirmed on the
basis of the liver biopsy findings, tetralogy of Fallot,
butterfly vertebrae, ocular findings and molecular
analysis (see below).
Patient B was born to a 33 year-old G2P0 Caucasian
woman who had 2 previous ectopic pregnancies but no
medical history of note. The parents were non-consan-
guinous. The pregnancy was unremarkable except for
mild pregnancy-induced hypertension. Patient B was
born at 42 weeks gestation via emergency cesarean
weight was 2.83 kg (10thcentile) and the length was
45 cm (<5thcentile). Patient B had mild transient
neonatal jaundice, which was not investigated. The
parents noticed an unusual appearance to the head
shape from birth.
At the age of 10 weeks persistent concern regarding
the head shape led to further investigation and the
diagnosis of left coronal synostosis was made. At this
time scleral icterus was also detected and the bilirubin
fraction. A liver biopsy was performed which revealed
paucity of bile ducts. No vertebral anomalies were
detected on X-ray and an opthalmological examination
was inconclusive. An echocardiogram revealed mild
peripheral pulmonary stenosis, pulmonary stenosis,
mild aortic stenosis and an atrial septal defect. The
diagnosis of AGS was made based on the liver biopsy
findings, facial features and the cardiac defect.
PatientBrequired corrective neurosurgeryand facial
further cardiac intervention and the atrial septal defect
our institution at 4 years of age. His clinical course had
largely been dominated by his hepatic disease. He has
had significant problems with pruritis and large xan-
a flattened tip, deep-set eyes, pointed chin—see Fig. 1A)
and scleral icterus. His head circumference was 48 cm
(<3rdcentile; 50thcentile for 18 months). The diagnosis
Craniosynostosis in Alagille Syndrome 177
of AGS was confirmed on the basis of the liver biopsy
findings, the cardiac defect, typical facial features and
molecular analysis (see below).
Mutational Analysis for JAG1
Genomic DNA was extracted using a commercially
available kit (Puregene DNA Isolation Kit, Gentra
Systems, Minneapolis, MN) according to the manufac-
turer’s instructions. Mutational analysis was perfor-
med by single stranded conformational polymorphism
(SSCP) analysis. All 26 exons were screened in 32
polymerase chain reactions as described previously
[Krantz et al., 1997]. In patient A, a shift in exon 16
was identified that upon sequencing revealed a 5 base
exon 13 was identified. Sequencing revealed a nonsense
mutation (E553X) (Fig. 2B).
Sequence Analysis of FGFR1, -2, -3 and TWIST
Genomic DNA was extracted using a commercially
available kit (Puregene DNA Isolation Kit, Gentra
Systems, Minneapolis, MN) according to the manufac-
turer’s instructions. Polymerase chain reaction amplifi-
cation of the TWIST gene was performed using
conditions and primers specific for the TWIST gene
[Howard et al., 1997]. Using techniques described pre-
was amplified and analysis was performed to detect the
C to G mutation that results in the Pro252Arg amino
acid substitution. Exons 8 and 10 of the FGFR 2 gene
were amplified using primers and conditions described
by [Meyers et al., 1996]. Exon 7 of the FGFR 3 gene was
[Bellus et al., 1996], the C to G mutation that results in
the Pro250Arg amino acid substitution was sought.
Sequencing of amplified DNA was performed using the
Perkin Elmer Applied Biosystems cycle sequencing kit
(Foster City, CA) according to manufacturer’s instruc-
tions. Sequences were analyzed on an automated DNA
sequencer (ABI 377, Foster City, CA). No mutations in
FGFR1,-2,-3 or TWIST were detected in either patient
A or B.
Both individuals identified had mutation proven AGS
and unilateral left coronal synostosis. Population based
estimates of craniosynostosis reveal a birth prevalence
of 343/1,000,000 (0.03%) for all craniosynostosis cases
syndromic coronal synostosis in the general population
is estimated as 94/1,000,000 (0.01%) [Lajeunie et al.,
1995]. We have examined 230 probands with AGS
through the Alagille Syndrome Diagnostic Center
(ASDC) at The Children’s Hospital of Philadelphia.
from this group suggests a prevalence of 1 in 115 (0.9%)
in this AGS population. Though the number of cases is
small this prevalence is significantly higher than in the
The molecular basis for many craniosynostosis syn-
dromes has been identified. Mutations in the FGFR1, -2
and -3 genes have been identified in the major craniosy-
nostosis syndromes [Cohen, 1995]. Fibroblast growth
factors (FGF) act as ligands at these receptor sites and
are thought to mediate signalling in the differentiation
tissues of the skull [Sarkar et al., 2001]. Fibroblast
growth factor receptor (FGFR) mutations cause some of
the well-known craniosynostosis syndromes such as
Apert syndrome and Crouzon syndrome. Despite the
molecular advances there remains significant clinical
of known mutations. In some cases the exact same mu-
tation may cause two different syndromes [Cohen and
Maclean, 2000]. In addition, different mutations may
sometimes cause the same syndrome. The role of addi-
tional modifying genes is not fully understood. Further-
more, there remain a significant number of patients
with non-syndromic craniosynostosis in whom no gene-
for a mutation in FGFR1 associated with Pfeiffer syn-
exon 16 was identified in patient A (A). Arrow indicates site of deletion.
Sequence is from a single standed amplification product. A nonsense
mutation in exon 13 (E553X) converting a G to a T at base 2070 (arrow) was
identified in patient B (B).
Results ofJagged1 mutational analysis. A 5base pair deletion in
178 Kamath et al.
drome and for mutations in FGFR2 known to cause
Pfeiffer, Crouzon and Jackson-Weiss syndromes. Of
note, a mutation has been identified in FGFR3, which is
associated with isolated unilateral coronal synostosis,
et al., 1998]. Sequence analysis was also performed for
this mutation and was negative. Further testing in the
regions of the TWIST gene known to contain mutations
causingSaethre-Chotzen syndrome was unremarkable.
The lack of identification of a mutation at one of the
known craniosynostosis loci in these AGS individuals
or predisposing role. It is worth considering, however,
known craniosynostosis loci and it is possible that they
may carry hitherto unknown mutations elsewhere in
the genome, other than JAG1, that contributed to the
This is the first report that suggests alterations in
the Notch signalling pathway cause craniosynostosis.
However there are previous reports indicating that
mutations in the FGF pathway can cause clinical
manifestations which have overlap with AGS. Ocular
et al., 1999]. The predominant abnormalities are
anterior chamber defects (posterior embryotoxon or
Axenfeldanomaly) [Puklinet al.,1981]. Ocular anterior
chamber abnormalities have been recently described in
craniosynostosis syndromes with a mutation in FGFR2
[Okajima et al., 1999]. Vertebral anomalies are also a
common finding in AGS, characteristically butterfly
syndrome, which can be caused by FGFR mutations,
also have abnormalities in the vertebrae such as
hemivertebrae and butterfly vertebrae [Anderson et al.,
1996], both of which are seen in AGS. Hence there is
evidence suggesting clinical overlap between the Notch
pathway and pathways involved in cranial suture
systems provide further evidence for interaction be-
tween Notch and cranial suture pathways. In mouse
neuroepithelial precursor cells data suggests the inhi-
bitory action of growth factors, such as FGF1 and 2, is
in mouse dental epithelium to determine stem cell fate
[Harada et al., 1999].
The mutations in JAG1 seen in the individuals
described in this report both result in truncated protein
products. These mutations (in exons 13 and 16) have
tions may play a contributory role in the development of
lack of genotype–phenotype correlation is generally
characteristic of JAG1 mutations.
If JAG1 is involved in cranial suture development, a
demonstrated JAG1 expression in the cardiovascular
system, limb buds, kidney, eye, ear and central nervous
system [Crosnier et al., 2000; Jones et al., 2000].
Investigators have not reported JAG1 expression in
cranial neural crest cells though specific studies to
determine expression in the developing cranium have
not been performed. Though liver disease is a major
cause of morbidity in AGS, JAG1 expression is only
detected in blood vessels in the liver, and not in devel-
JAG1 mutations can predispose to craniosynostosis via
its profound effects in the vascular system.
We propose that JAG1 and the Notch signalling
pathway may play a role in cranial suture development
and mutations in the gene predispose to craniosynos-
tosis. The above normal prevalence of a conserved form
loci supports the hypothesis. Furthermore, the identifi-
cation of craniosynostosis patients with ocular anterior
chamber defects and vertebral anomalies and now the
finding of craniosynostosis in AGS patients, suggests
that JAG1 and the Notch signalling pathway may
interact with pathways that are important in cranial
suture development, such as FGF pathways. Evidence
from mammalian systems further supports the interac-
tion. This is the first report of craniosynostosis in
individuals with AGS and the finding indicates that
JAG1and theNotch signalling pathway mayplay arole
in cranial suture development.
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