American Journal of Medical Genetics 112:163– 170 (2002)
Facial Features in Alagille Syndrome: Specific
or Cholestasis Facies?
Binita M. Kamath,1 Kathleen M. Loomes,1 Rebecca J. Oakey,2 Karan E.M. Emerick,3
Tracy Conversano,2 Nancy B. Spinner,2 David A. Piccoli,1 and Ian D. Krantz2*
1
Division of Gastroenterology and Nutrition, The Children’s Hospital of Philadelphia and University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania
2
Division of Human Genetics and Molecular Biology, The Children’s Hospital of Philadelphia and University
of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
3
Division of Gastroenterology, Children’s Memorial Hospital, Chicago, Illinois
Alagille syndrome is a complex multisystem
disorder characterized by bile duct paucity,
cholestasis, cardiac defects, vertebral ano-
malies, ophthalmologic changes, and facial
dysmorphism. Although the facial features
are highly conserved in affected individuals
both within and between families, the possi-
bility has been raised that cholestasis is the
causative factor for the facies. In this study,
the diagnostic specificity of the facies in Ala-
gille syndrome has been evaluated by asking
clinical dysmorphologists to examine a pho-
tographic panel of 18 pediatric and adult
individuals with Alagille syndrome and
other forms of congenital intrahepatic cho-
lestasis. The examiners had no knowledge of
the actual diagnoses. The group was able to
distinguish correctly between Alagille and
non-Alagille individuals with a frequency of
79%. Professional grade of the respondent
did not affect the accuracy of correct identi-
fication. The adult facial features were the
most difficult to evaluate successfully. The
sensitivity of facies identification to diag-
nose Alagille syndrome was 76%, the specifi-
city 82%, the positive predictive value 81%,
and the negative predictive value 77%. These
results suggest that the facies seen in Ala-
gille syndrome is specific to this condition
Grant sponsor: NIDDK; Grant numbers: 1K08 DK02541-01 (to
I.D.K.), 1 K08 DK02 796-01CK, 1R01 DK53104 (to N.B.S.); Grant
sponsor: The Fred and Suzanne Biesecker Center for Pediatric
Liver Disease at The Children’s Hospital of Philadelphia.
*Correspondence to: Dr. Ian D. Krantz, 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.
E-mail: ian2@mail.med.upenn.edu
Received 29 June 2001; Accepted 4 February 2002
DOI 10.1002/ajmg.10579
ß 2002 Wiley-Liss, Inc.
and its recognition is a valuable tool in
diagnosis. ß 2002 Wiley-Liss, Inc.
KEY WORDS: Alagille syndrome; cholesta-
sis; facies; dysmorphology;
Jagged1
INTRODUCTION
Alagille syndrome (AGS; OMIM 118450) is a complex
dominantly inherited multisystem disorder involving
predominantly the liver, heart, eyes, face, and skeleton
[Krantz et al., 1999a]. The facial dysmorphism described
in AGS consists of a prominent forehead, deep-set eyes
that may appear or be hyperteloric, a straight nose with
a flattened tip, and a prominent and in some cases
pointed chin (Fig. 1). Due to the constellation of these
features, the overall appearance of the face is of an
inverted triangle. Classically, the diagnosis of AGS has
been based on clinical criteria set forth by Alagille et al.
[1975]. The clinical criteria for diagnosis include the
histological finding of bile duct paucity on liver biopsy in
association with three out of five major clinical features
(cholestasis, cardiac defects, vertebral anomalies, oph-
thalmologic findings, and facial features). Presence of
the facies is considered a significant clinical finding and
is frequently integral to making the diagnosis. In a
review of 80 cases, Alagille et al. [1987] determined the
facial phenotype to be present in 95% of cases. This has
been further supported by Emerick et al. [1999], who
found this frequency to be 96% in a series of 92 patients.
Though the frequent presence of a typical facial phe-
notype in AGS has been well accepted, the cause and
specificity of the facies have been a subject of debate. It
has been proposed by Sokol et al. [1983] that the facial
dysmorphism seen in AGS is nonspecific and secondary
to congenital intrahepatic cholestasis from a variety of
causes. They describe a ‘‘cholestasis facies’’ that is either
the result of a common structural effect on facial devel-
opment by involved disease genes or due to the effect of
the multiple biochemical aberrations caused by the
cholestasis itself.
164 Kamath et al.
Fig. 1. Typical facial features of Alagille syndrome in a 8-year-old child.
Note the prominent forehead, deep-set eyes, bulbous tip of the nose, and
sharply pointed chin.
The clinical experience of our group does not support
the idea of a common ‘‘cholestasis facies,’’ and there are
data to support the hypothesis of a facies specific to AGS.
Jagged1 (JAG1) has been identified as the disease gene
for AGS [Li et al., 1997]. JAG1 is a ligand in the Notch
Fig. 2. Pedigree showing mutation-positive family members sharing typical facial features of AGS. However, the affected daughter (lower right) has had
no clinical or biochemical evidence of cholestasis.
signaling pathway and plays a role in early cell fate
determination. In situ studies to determine the expres-
sion pattern of JAG1 in developing mouse and human
embryos show a specific pattern of JAG1 expression in
the facial structures. Further evidence in favor of a
facies specific to AGS is the identification of family
members of mutation-positive probands, who have par-
tial phenotypes of AGS and have the characteristic
facies without liver involvement (Fig. 2).
No other study has been undertaken since the original
study by Sokol et al. [1983] to assess the diagnostic
specificity of the facies in AGS. Evaluating the recogni-
tion of facial features is a difficult subject of study and
the original work was limited in a number of areas. Only
13 examiners evaluated the facial features in the
original cohort of Sokol et al. [1983] and the examiners
were hepatologists, not trained dysmorphologists.
Furthermore, the diagnoses of the AGS individuals exa-
mined were not confirmed by mutational analysis, as the
disease gene had not yet been identified at that time.
This study aims to assess more accurately the specificity
of the facial dysmorphism in AGS and the value of facies
recognition as a diagnostic tool.

MATERIALS AND METHODS
Questionnaire
A photographic panel was created of 18 subjects,
which included 9 individuals with AGS and 9 with
congenital intrahepatic cholestasis caused by known
diagnoses other than AGS. All diagnoses were estab-
lished using standard clinical and histological criteria.
In addition, all AGS patients had known mutations in
JAG1. Of the 18 patients, 9 had mutation-proven AGS
and of the 9 non-AGS controls, 4 had biliary atresia and 5
had alpha-1-antitrypsin deficiency. The panel included
a sampling of patients from various ages, ethnic back-
grounds, and gender in both the AGS and non-AGS
matched controls and were randomly selected over a
Fig. 3. Full-face and profile views of the individuals included in the photographic panels distributed to the dysmorphologists participating in this study.
All photographs were gray-scale so that the reviewers would not be influenced by the presence or absence of jaundice. Original dimensions of all individual
images were 8
7.5 cm.
Facial Features in Alagille Syndrome 165
period of 1 year from patients seen at the Children’s
Hospital of Philadelphia so as not to reflect a selective
bias on the part of the authors. All patients were pho-
tographed full face and in profile and the pictures were
displayed in black and white (Fig. 3). Informed consent
was obtained from all subjects to take and use the
photographs in the study.
The mixed photographic panel was contained within a
questionnaire, which included a brief clinical descrip-
tion of AGS, some background to the study, and a score
sheet (see appendix online). Two 8 by 7.5 cm black-and-
white pictures of each individual were displayed (full
face and profile), with three cases per page. The ques-
tionnaire also collected information about the position
and title of the respondent. The questionnaire was
166 Kamath et al.
accompanied by a poster presentation about facial dys-
morphism in AGS. Participants of the 1998 David W.
Smith Annual Workshop on Malformations and Mor-
phogenesis were requested to score the photographs on
the basis of facial features as to whether the individuals
in the photographs did or did not have AGS. The parti-
cipants had no knowledge of the actual hepatic diagnos-
es. A positive response was one in which the respondent
correctly identified the presence or absence of AGS.
The following variables were calculated using stan-
dard statistical methods to evaluate recognition of facial
features as a tool for diagnosing AGS: the sensitivity (the
percentage of positive identifications from the group
with AGS), the specificity (the percentage of negative
identifications from the group with cholestasis and
without AGS), the positive predictive value (the percen-
tage of positive identifications that were correct diag-
noses of AGS), and the negative predictive value (the
percentage of negative identifications that correctly do
not have AGS) of the test.
Mouse In Situ Hybridization Studies
Tissue collection and preparation. CD1 mouse
embryos (Charles River Laboratory) were harvested
from timed matings at 10.5 and 12.5 d.p.c. Embryos for
sectioning were fixed overnight in 4% paraformalde-
hyde, dehydrated, and embedded in paraffin wax.
Embryos to be used for whole mount in situ hybridiza-
tion were fixed overnight in 4% paraformaldehyde,
dehydrated, and stored in methanol at 48C.
Section in situ hybridization. Sense (control)
and antisense (test) riboprobes were transcribed from
linearized DNA templates in the presence of 35[S]UTP
using an in vitro transcription kit (Roche, Indianapolis,
IN). Probe templates were as follows. Mouse JAG1
probe: EST (expressed sequence tag; Research Genetics,
Huntsville, AL) containing 2.2 kb of mouse JAG1,
Fig. 4. JAG1 is expressed in primordial facial structures in the
developing mouse embryo. A: Whole mount in situ hybridization using a
probe for JAG1 on a 10.5 d.p.c. mouse embryo shows prominent expression in
first pharyngeal arch (1st PrA, arrow). The 1st PrA will give rise to facial
structures, including the maxilla and the mandible. JAG1 expression is also
demonstrated in the eye (E), otic pit (OP), and heart (H). Gene expres-
sion¼purple. B: Section in situ hybridization on a 12.5 d.p.c. mouse embryo
including the polyA tail and 30 UTR, cytoplasmic domain,
and part of the EGF repeats [Loomes et al., 1999]. The
antisense template was linearized with EcoRI and
transcribed with T3 polymerase. The sense template
was linearized with NotI and transcribed with T7 poly-
merase. The general protocol for section in situ hybrid-
ization and probe labeling is essentially as in Sassoon
and Rosenthal [1993] and Wilkinson and Nieto [1993].
Whole mount in situ hybridization. Mouse em-
bryos were fixed overnight in 4% paraformaldehyde and
taken through a series of washes. The general protocol
for whole mount in situ hybridization is essentially as in
Rosen and Beddington [1993] and Wilkinson and Nieto
[1993]. Probe templates were as described above. Digo-
xigenin-labeled sense and antisense riboprobes were
transcribed from linearized DNA templates using an
in vitro transcription kit (Roche, Indianapolis, IN).
RESULTS
In Situ Hybridization Studies
At 10.5 days d.p.c. in the mouse embryo, JAG1
expression is detected in the first pharyngeal arch
(Fig. 4A, arrow). The first pharyngeal arch will give rise
to facial structures including the maxilla and the man-
dible. Later, in mid-gestation at 12.5 d.p.c. in the mouse
embryo, JAG1 is expressed in several regions in the
head and neck, including the nasopharynx, the tongue,
and the mandible (Fig. 4B). Similar patterns of JAG1
expression are seen in the primordial facial structures in
human embryos (data not shown) [Crosnier et al., 2000;
Jones et al., 2000].
Facial Dysmorphism Analysis
Forty-nine individuals participated in the study. The
respondents were all actively working in clinical gene-
tics and included 5 fellows, 10 assistant professors, 11
shows JAG1 expression in facial structures, including the nasopharynx (NP,
arrow), the tongue (T), and the mandible (M). JAG1 is also expressed in the
cells lining the ventricles (V) and in the cardiac outflow tract (OFT, arrow).
Gene expression ¼ yellow. Scale bar in A for A and B ¼ 300mM. [Color figure
can be viewed in the online issue, which is available at www.interscience.-
wiley.com]
 Facial Features in Alagille Syndrome 167

associate professors, 16 full professors, and 7 others
(including clinical consultants, private practitioners,
and staff physicians). The correct identification of AGS
versus non-AGS based on the photographs ranged from
41% to 100% (Table I).
Overall, the 49 individuals were able to identify the
AGS facies 79% of the time. The percentage of correct
identifications did not appear to be affected by the pro-
fessional grade of the respondent: fellows 80%, assistant
professors 82%, associate professors 79%, full professors
77%, and others 79% (Table II).
The calculated sensitivity, that is, the likelihood of
positive facies recognition in the presence of AGS, was
76%. The specificity, that is, the likelihood of not iden-
tifiying AGS facial features in individuals with con-
genital cholestasis who do not have AGS, was 82%. The
positive predictive value, that is, the probability that an
individual has AGS given that he or she has a positive
screening result (presence of facial features), was 81%.
The negative predictive value, that is, the probability
that an individual does not have AGS given that he or
she has a negative screening result (absence of facial
features), was 77%. It is of note that the facial features of
the adult individual with AGS appeared to be the most
difficult to identify accurately (identified only 67% of the
time compared to 78% of the infants; Table III).
DISCUSSION
This study demonstrates the ability of clinical dys-
morphologists to distinguish between AGS and non-
AGS patients based solely on evaluation of facial
features. These results add to the body of evidence sup-
porting the hypothesis of a specific facies in AGS. JAG1
expression has been demonstrated in the facial struc-
tures of developing mouse (Fig. 4) and human embryos
[Crosnier et al., 2000; Jones et al., 2000]. Family studies
have identified relatives of mutation-positive pro-
bands with no clinical or biochemical evidence of hepatic
abnormalities yet having the recognizable facial pheno-
type. The presence of the characteristic facies in such
individuals could not be secondary to cholestasis. These
data in combination imply that the AGS facies is specific
to the syndrome and not a common endpoint of cho-
lestasis as previously suggested. Identification of the
facial phenotype was shown to be a sensitive and specific
tool in the diagnosis of AGS.
These results contrast with the original work of Sokol
et al. [1983] in which examiners were able to distinguish
correctly between AGS and non-AGS on photographic
evaluation with a frequency of only 50% on average. The
sensitivity and specificity of facies as a diagnostic tool
were also lower than in our study. The increased accu-
racy in identification in this study may have arisen due
to the more skillful judgement of dysmorphologists in
evaluating facial features. Furthermore, since the study
by Sokol et al. [1983], the identification of JAG1 as the
AGS disease gene enabled us to confirm the diagnosis of
AGS on a molecular level in all of the participants being
studied in this report.
Both this and the study by Sokol et al. [1983] are
limited by attempting to assess morphology from two-
dimensional photographs. Facial features may be more
noticeable in person or on video display. Despite a larger
number of examiners in this study compared to the
original, our work is similarly limited by a small number
of photographed individuals.
The difficulty demonstrated by the dysmorphologists
participating in this study in recognizing the adult AGS
facies supports the clinical experience of the authors
that several children with AGS are being called
‘‘isolated’’ based on studies of parents who may not have
other clinical features of AGS but have typical adult
facial features. Similarly, adult individuals being follow-
ed by cardiology who have structural heart defects but
no overt liver involvement who have typical adult AGS
facies and are at a 50% risk of passing the altered JAG1
gene to their offspring have been identified [Krantz et al.,
1999b; Eldadah et al., 2001]. The child’s facies in AGS
typically has a prominent forehead and delicate pointed
chin, giving the face an inverted triangular appearance.
At about the time of puberty, the chin becomes more

TABLE I. Number of Correct Answers for Each Face Reviewed

Face Assist Associate Full

number Fellow professor professor professor Other Total

1 5/5 10/10 11/11 16/16 7/7 49/49 (100%)

2 0/5 5/10 3/11 6/16 5/7 19/49 (39%)
3 5/5 10/10 9/11 14/16 6/7 44/49 (90%)
4 4/4 8/10 8/11 14/16 6/7 40/49 (82%)
5 5/5 10/10 11/11 16/16 7/7 49/49 (100%)
6 5/5 8/10 11/11 15/16 7/7 46/49 (94%)
7 5/5 10/10 10/11 12/16 7/7 44/49 (90%)
8 5/5 10/10 11/11 16/16 7/7 49/49 (100%)
9 2/5 5/10 4/11 9/16 6/7 26/49 (53%)
10 3/5 7/10 7/11 12/16 2/7 31/49 (63%)
11 5/5 9/10 10/11 10/16 5/7 39/49 (80%)
12 3/5 9/10 7/11 14/16 4/7 37/49 (76%)
13 1/5 4/10 5/11 5/16 5/7 20/49 (41%)
14 4/5 7/10 9/11 10/16 4/7 34/49 (70%)
15 5/5 9/10 11/11 10/16 4/7 39/49 (80%)
16 5/5 10/10 11/11 15/16 6/7 47/49 (96%)
17 5/5 9/10 9/11 12/16 5/7 40/49 (82%)
18 5/5 9/10 10/11 14/16 6/7 44/49 (90%)
168 Kamath et al.

TABLE II. Accuracy of Facial Diagnosis by Professional Group
Total number Range of
Professional group in group scores Average score
Fellows 5 13/18–16/18 14.4/18 (80%)
Assistant professor 10 13/18–18/18 14.8/18 (82%)
Associate professor 11 12/18–16/18 14.3/18 (79%)
Full professor 16 10/18–16/18 13.8/18 (77%)
Other 7 12/18–17/18 14.3/18 (79%)
Total 14.32/18 (80%)
prominent, becoming prognathic, and there is less domi-
nance of the forehead. The eyes remain deep-set. This
process almost inverts the childhood facial appearance
with a now predominant lower face and mandible and
less prominent upper face and forehead (Fig. 5).
Although pictorial representations of the adult AGS
facies have been extensively published [Alagille et al.,
1987; Mueller, 1987; Keeffe et al., 1993; Spinner et al.,
1994], the typical face that is familiar to dysmorpholo-
gists is that of the facies in infancy and childhood (not
unusual given that most of the complications encoun-
tered in individuals with AGS present during the
newborn period or shortly thereafter). Little attention
has been given to the adult AGS facies and the tran-
sitions that occur from childhood. The importance of
identifying the adult facies lies in the recognition of
subtle familial versus isolated cases of AGS as well as
individuals who may be followed for apparently isolated
congenital heart disease but may be at risk for having
severely affected children with full clinical manifesta-
tions of AGS. The recurrence risk of congenital cardiac
abnormalities in children of adults with truly isolated
cardiac defects is low, generally less than 5% [Nora and
Nora, 1976]. However, this risk rises to 50% in AGS, as
in other dominant conditions. If the adult facies could be
more readily identified, such individuals could be pro-
vided with more appropriate genetic counseling. To this
end, further studies are underway to quantify objec-
tively the facial features in childhood and adulthood
through anthropometric measurements to identify spe-
cific changes with age.
In summary, the facial phenotype in AGS is specific to
the syndrome and this study validates the importance of
facies recognition as a tool for diagnosis.
ACKNOWLEDGMENTS
We thank all of the families that participated in these
studies and the support of the Alagille Syndrome
Alliance. Supported by the National Institutes of Health
(NIDDK) 5 K08 DK02541-02 (to I.D.K.) and the National
Institutes of Health P50HL6217 (to R.J.D. and N.B.S.).
APPENDIX
Alagille syndrome (AGS;OMIM 118450) is a complex
dominantly inherited multisystem disorder involving
the liver, heart, eyes, facies, skeleton, and other systems.
The facial dysmorphism described in AGS consists of a
prominent forehead, deep-set eyes that may appear or be
hyperteloric, a straight nose with a flattened tip, and a
prominent and in some cases pointed chin. Due to the
constellation of these features, the overall appearance of
the face is of an inverted triangle.
There has been debate among clinicians as to the
etiology of the facies seen in AGS. It has been proposed
by Sokol et al. [1983] that the facial dysmorphism seen in
AGS is nonspecific and is a result of congenital
intrahepatic cholestasis. They argue that the facies in
several conditions of congenital cholestasis are the
result of a common structural effect on facial develop-
ment by involved disease genes, or due to the effect of the
multiple biochemical aberrations caused by the chole-
stasis itself. No other study has been undertaken since
the original study by Sokol et al. [1983], and none at all
by clinical dysmorphologists to assess the diagnostic
specificity of the facies in AGS.
We are undertaking a systematic approach to study-
ing the facial dysmorphism in AGS. Jagged1 has
recently been identified to be the disease gene for AGS.
Jagged1 is a ligand in the Notch signaling pathway and
plays a role in early cell fate determination. Studies are
underway to determine its expression pattern in devel-
oping human and mouse embryos, with an emphasis on
the facial structures. Preliminary studies show positive
expression in the facial structures. We are also evaluat-
ing family members of mutation-positive AGS probands
who have partial phenotypes of AGS and have found
several individuals who have facies without liver
involvement. These data in conjunction with our clinical
experience have led us to believe that the Alagille facies
are specific to this syndrome and not the result of a
nonspecific intrauterine cholestatic effect.
In order to have an unbiased evaluation of the facial
dysmorphism, we have accumulated a mixed photo-
graphic panel of 18 patients either with AGS (all with
identified Jagged1 mutations) or with congenital intra-
hepatic cholestasis caused by known diagnoses other
than AGS. All patients were photographed full-face and
in profile. Based on your past experience, and on the
information provided here and in the associated poster,
please attempt to score the photos of the patients as

TABLE III. Accuracy of Facial Diagnosis by Patient Age

Number of Assistant Associate Full

Age group patients Fellows professor professor professor Other Total

0–24 months 3/18 80% 83% 76% 83% 67% 78%

24–60 months 3/18 67% 80% 67% 63% 76% 71%
5–10 years 6/18 83% 90% 83% 90% 88% 87%
11–16 years 4/18 95% 83% 91% 73% 71% 83%
Adults 2/18 60% 70% 68% 53% 86% 67%

Fig. 5. Photographs of the facial features of an individual with AGS at infancy, adolescence, and adulthood demonstrating the evolution of the facial
features.
having Alagille syndrome or not on the attached score
sheet.
We have attempted to provide a sampling of patients
from various ages, ethnic backgrounds, and gender in
both the AGS and non-AGS matched controls.
Thank you for taking the time to complete this
questionnaire. It will prove invaluable to the completion
of this study.
Questionnaire
Facial Features in Alagille Syndrome 169
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