Spectrum of JAG1 gene mutations in Polish patients with Alagille syndrome

Abstract

Alagille syndrome (ALGS) is an autosomal dominant disorder characterized by developmental abnormalities in several organs including the liver, heart, eyes, vertebrae, kidneys, and face. The majority (90-94 %) of ALGS cases are caused by mutations in the JAG1 (JAGGED1) gene, and in a small percent of patients (∼1 %) mutations in the NOTCH2 gene have been described. Both genes are involved in the Notch signaling pathway. To date, over 440 different JAG1 gene mutations and ten NOTCH2 mutations have been identified in ALGS patients. The present study was conducted on a group of 35 Polish ALGS patients and revealed JAG1 gene mutations in 26 of them. Twenty-three different mutations were detected including 13 novel point mutations and six large deletions affecting the JAG1 gene. Review of all mutations identified to date in individuals from Poland allowed us to propose an effective diagnostic strategy based on the mutations identified in the reported patients of Polish descent. However, the distribution of mutations seen in this cohort was not substantively different than the mutation distribution in other reported populations.

Full text

HUMAN GENETICS • ORIGINAL PAPER
Spectrum of JAG1 gene mutations in Polish patients
with Alagille syndrome
Dorota Jurkiewicz & Dorota Gliwicz & Elżbieta Ciara & Jennifer Gerfen &
Magdalena Pelc & Dorota Piekutowska-Abramczuk & Monika Kugaudo &
Krystyna Chrzanowska & Nancy B. Spinner & Małgorzata Krajewska-Walasek
Received: 8 January 2014 /Revised: 19 March 2014 / Accepted: 27 March 2014 /Published online: 20 April 2014
#
The Author(s) 2014. This article is published with open access at Springerlink.com
Abstract Alagille syndrome (ALGS) is an autosomal domi-
nant disorder characterized by developmental abnormalities in
several organs including the liver, heart, eyes, vertebrae, kid-
neys, and face. The majority (90-94 %) of ALGS cases are
caused by mutations in the JAG1 (JAGGED1)gene,andina
small percent of patients (∼1 %) mutations in the NOTCH2
gene have been described. Both genes are involved in the
Notch signaling pathway. To date, over 440 different JAG1
gene mutations and ten NOTCH2 mutations have been iden-
tified in ALGS patients. The present study was conducted on a
group of 35 Polish ALGS patients and revealed JAG1 gene
mutations in 26 of them. Twenty-three different mutations
were detected including 13 novel point mutations and six large
deletions affecting the JAG1 gene. Review of all mutations
identified to date in individuals from Poland allowed us to
propose an effective diagnostic strategy based on the muta-
tions identified in the reported patients of Polish descent.
However, the distribution of mutations seen in this cohort
was not substantively different than the mutation distribution
in other reported populations.
Keywords Alagille syndrome
.
Diagnostic strategy
.
JAG1
gene
.
JAG1 point mutations
.
Large deletions
Introduction
Alagille syndrome (ALGS, OMIM #118459) is an autosomal
dominant disorder characterized by developmental abnormal-
ities in several organs including the liver, heart, eyes, verte-
brae, kidneys, and face (Alagille et al. 1987). The diagnosis of
ALGS is based on the appearance of bile duct paucity with at
least three of the major clinical features including: chronic
cholestasis, cardiac disease, skeletal abnormalities, ocular ab-
normalities, renal anomalies, and characteristic facial features
(Emerick et al. 1999). The diagnosis of ALGS is hampered by
its highly variable expressivity despite almost complete pen-
etrance (Dhorne-Pollet et al. 1994). ALGS is caused by mu-
tations in the JAG1 (JAGGED1; MIM# 601920) or the
NOTCH2 (MIM# 600275) genes (Li et al. 1997; Oda et al.
1997; McDaniell et al. 2006;Kamathetal.2012). Both genes
are involved in the Notch signaling pathway. The JAG1 gene
encodes a cell surface ligand, whereas the NOTCH2 gene
encodes one of the four human Notch receptors. The JAG1
gene is located within chromosome 20p12 and contains 26
exons encoding a conserved transmemb rane protein. The
JAG1 protein contains several evolutionarily conserved mo-
tifs, including a signal peptide, a DSL domain (Delta/Serrate/
Lag2), 16 epidermal growth factor (EGF)-like repeats, a
cysteine-rich region (CR), and a transmembrane domain
(Lindsell et al. 1995). Mutations in the JAG1 can be identified
in around 90 % of clinically diagnosed individuals with ALGS
(Warthen et al. 2006). To date, over 440 different JAG1
gene mutations have been identified in ALGS patients
D. Jurkiewicz (*)
:
E. Ciara
:
M. Pelc
:
D. Piekutowska-Abramczuk
:
M. Kugaudo
:
K. Chrzanowska
:
M. Krajewska-Walasek
Department of Medical Genetics, The Children’s Memorial Health
Institute, Al. Dzieci Polskich 20, 04-730 Warsaw, Poland
e-mail: d.jurkiewicz@czd.pl
D. Gliwicz
Department of Gastroenterology, The Children’s Memorial Health
Institute, Warsaw, Poland
J. Gerfen
:
N. B. Spinner
Department of Pathology and Laboratory Medicine, The Children’s
Hospital of Philadelphia and The Perelman School of Medicine,
The University of Pennsylvania, Philadelphia, PA, USA
M. Kugaudo
Department of Child and Adolescent Psychiatry, Medical University
of Warsaw, Warsaw, Poland
J Appl Genetics (2014) 55:329–336
DOI 10.1007/s13353-014-0212-2

(Li et al. 1997; Oda et al. 1997;Krantzetal.1998;Yuanetal.
1998, 2001;Crosnieretal.1999, 2000; Onouchi et al. 1999;
Piliaetal.1999; Heritage et al. 2000, 2002;Collitonetal.
2001; Giannakudis et al. 2001; Röpke et al. 2003;Jurkiewicz
et al. 2005; Warthen et al. 2006;Kamathetal.2009;Guegan
et al. 2012; Lin et al. 2012;Wangetal.2012). T en individuals
with ALGS features carrying various mutations in the
NOTCH2 gene have been reported to date (McDaniell et al.
2006;Kamathetal.2012).
The purpose of this study was to determine the spectrum of
JAG1 mutations in a group of 35 Polish ALGS patients. The
additional aim of the study was to review all mutations iden-
tified so far in Polish patients (Giannakudis et al. 2001;
Stankiewicz et al. 2001; Röpke et al. 2003; Jurkiewicz et al.
2005, 2006) and compare them with mutations described in
ALGS patients from other populations.
Materials and methods
Patients
Molecular analysis was performed in a group of 35 patients.
The group consisted of 22 new unrelated patients referred to
the Medical Genetics Laboratory in the Children’sMemorial
Health Institute (CMHI) who have not been reported before
and 13 patients from 11 unrelated families referred to CMHI
in whom JAG1 mutations were not revealed in previous
studies. The group of the patients without detected JAG1
mutations was originally reported (Jurkiewicz et al. 2005,
2006) and has been included in the present study after re-
evaluation of clinical data. The patients who had three or more
of the major clinical features of ALGS were referred for
genetic testing. All patients met the standard clinical diagnos-
tic criteria for ALGS (Alagille et al. 1987), although not all
individua ls h ad a liver biopsy performed. The study was
approved by the Bioethics Committee of the Children’s
Memorial Health Institute in Warsaw. Informed consent was
obtained from all participating patients and their legal
representatives.
Mutation detection and analysis
Blood samples were collected from patients and their family
members and genomic DNA was extracted from peripheral
blood leukocytes by use of standard procedures. The complete
coding sequence of the JAG1
gene (26 exons) was amplified
by polymerase chain reaction (PCR) as previously described
(Krantz et al. 1998; Colliton et al. 2001; Warthen et al. 2006).
PCR products were evaluated by a combination of single
strand conformation polymorphism (SSCP) analysis, which
was carried out on a GenePhor system (GE Healthcare, UK)
and bi-directional sequencing on an ABI 3130 or an ABI 3730
analyzer (Applied Biosystems, Foster City, CA, USA).
Sequences of analyzed fragments were compared with the
JAG1 cDNA sequence (GenBank RefSeq: NM_000214.2).
The numbering of the nucleotide changes that were revealed
was based on the reference sequence with the A of the ATG
translation initiation codon as nucleotide +1.
ALGS patients found to be negative for JAG1 mutations by
sequencing were screened for large deletions by multiplex
ligation probe-dependent amplification (MLPA) using the
SALSA MLPA kit P184 JAG1 (MRC-Holland, Amsterdam,
the Netherlands) according to the manufacturer’sinstructions.
MLPA was performed with 200 ng of genomic DNA. Probe
amplification products were run on the ABI 3730 DNA ana-
lyzer. Peak plots were visualized and normalized, and the
dosage ratios were calculated using GeneMarker software
v1.8 (Soft Genetics LLC, State Collage, PA, USA). Probe
ratios below 0.67 were considered to indicate a deletion and
if the ratio was above 1.33, a duplication was called. Samples
from healthy control subjects were included in each assay.
Analysis of samples showing evidence of alterations was
repeated three times.
In three patients with MLPA alterations chromosomal mi-
croarray analysis was carried out using the Illumina Infinium
SNP genotyping platform (Kamath et al. 2009). For other
patients with abnormal MLPA aCGH analysis was performed
using CGX3×720K or Human CGH 3×1.4 M WG v.1.0
arrays (Roche NimbleGen, Madison, WI, USA) according to
the manufacturer’s instructions, data were analyzed using
DEVA a nd Genoglyphix software (Roche NimbleGen,
Madison, WI, USA). The patients analyzed with the SNP
array were examined again with Roche NimbleGen arrays
for consistency of results.
In five patients without JAG1 mutations or large JAG1
deletions, the 34 exons of the NOTCH2 gene were sequenced
using 36 primer pairs (McDaniell et al. 2006). The sequences
generated were compared with the NOTCH2 cDNA sequence
(GenBank RefSeq: NM_024408).
DNA samples obtained from additional family members
were screened following identification of a JAG1 mutation in
the proband.
The results from the analysis of the patients described in
this study were then combined with results from JAG1 anal-
ysis of 30 additional Polish patients previously reported in the
literature (Giannakudis et al. 2001; Stankiewicz et al. 2001;
Röpke et al. 2003; Jurkiewicz et al. 2005, 2006).
Results
Mutations in the JAG1 gene were found in 26 of the 35 ALGS
patients (Table 1). Mutations were identified in 18 of the 22
newly studied patients, and eight of the 13 patients that had
been previously screened by SSCP. Twenty-three different
330 J Appl Genetics (2014) 55:329–336

Table 1 The JAG1 gene mutations in Polish ALGS patients. All mutations identified in the current study as well as mutations detected in previous
studies (Giannakudis et al. 2001; Stankiewicz et al. 2001; Röpke et al. 2003; Jurkiewicz et al. 2005, 2006) are shown
Patient
No.
Exon or intron Mutation position
a
Predicted
consequence
Protein domain
b
Origin
c
Phenotype
d
Reference
Frameshift 1 Ex 2 c.172_178del7 p.(Ala58fs) SP-DSL NM L, H, E, F Jurkiewicz et al. (2005)
2 Ex 4 c.509delT p.(Leu170fs) SP-DSL NM L, H, E, F Jurkiewicz et al. (2005)
3 Ex 7 c.929delG p.(Gly310fs) EGF3 de novo L, E, V, F This study
4 Ex 9 c.1197delG p.(Val399fs) EGF5 de novo L, H, F Jurkiewicz et al. (2005)
5 Ex 12 c.1456_1457delAG p.(Arg486fs) EGF7 de novo L, H, E, F Jurkiewicz et al. (2006)
6 Ex 12 c.1485_1486delCT p.(Pro495fs) EGF8 de novo L, H, E, V, F Jurkiewicz et al. (2005)
7Ex14 c.1736_1737delCA p.(Thr579fs) EGF10 de novo L, H, E, V
?
, F This study
8 Ex 14 c.1809_1810insTGGG p.(Lys604fs) EGF10 maternal L, H ,E, F Jurkiewicz et al. (2005)
9Ex15 c.1897delT p.(Cys633fs) EGF11 de novo L, H, V, F, R This study
10 Ex 16 c.2065_2066delTT p.(Phe689fs) EGF12 de novo L, H, E, F Röpke et al. (2003)
11 Ex 17 c.2122_2125delCAGT p.(Gln708fs) EGF13 ND L, H, E, F Jurkiewicz et al. (2005)
12 Ex 18 c.2250delC p.(Pro750fs) EGF14 de novo L, H, V, F Röpke et al. (2003)
13 Ex 22 c.2648delG p.(Cys883fs) CR de novo L, H, E, V, F This study
14 Ex 22 c.2651-2652insA p.(Gln884fs) CR de novo L, H, E, F This study
15 Ex 23 c.2753delT p.(Ile918fs) CR NM L, H, E, F Jurkiewicz et al. (2005)
16 Ex 25 c.3197_3198insC p.(Thr1066fs) CR-TM ND L, H, E, V
?
, F, R This study
17 Ex 26 c.3230_3231insT p.(Leu1077fs) TM ND L, H, E, V, F This study
Nonsense 18 Ex 2 c.142G>T p.(Glu48Ter) SP-DSL paternal L, H, E, F Jurkiewicz et al. (2006)
19 Ex 2 c.246T>G p.(Tyr82Ter) SP-DSL paternal L, H, E, V, F This study
20 Ex 2 c.383G>A p.(Trp128Ter) SP-DSL de novo L, H, F Jurkiewicz et al. (2005)
21 Ex 4 c.496C>T p.(Gln166Ter) SP-DSL de novo L, V, F Jurkiewicz et al. (2005)
22 Ex 5 c.703C>T p.(Arg235Ter) EGF1 de novo L, H, E, V, F This study
g
23 Ex 6 c.841C>T p.(Gln281Ter) EGF2 paternal L, H, E, V, F Jurkiewicz et al. (2005)
24 Ex 9 c.1207C>T p.(Gln403Ter) EGF5 de novo L, H, F Jurkiewicz et al. (2005)
25 Ex 10 c.1325G>A p.(Trp442Ter) EGF6 maternal L, H, E, V, F This study
g
26 Ex 13 c.1603C>T p.(Gln535Ter) EGF9 de novo L, H, E, F Jurkiewicz et al. (2005)
27 Ex 18 c.2230C>T p.(Arg744Ter) EGF14 de novo L, H, E, V, F This study
28 Ex 18 c.2304C>A p.(Cys768Ter) EGF14 maternal L, V, F This study
Splice site 29 IVS 2 c.388-1G>C r.spl? SP-DSL de novo L, H, E, F Jurkiewicz et al. (2005)
30 IVS 3 c.439+1G>A r.spl? SP-DSL maternal L, H, E, V, F Jurkiewicz et al. (2006)
31 IVS 3 c.439+1G>A r.spl? SP-DSL de novo L, H, E, F, R Jurkiewicz et al. (2006)
32 IVS 5 c.755+1G>A r.spl? EGF1 maternal L, H, E, V, F Giannakudis et al. (2001)
33 IVS 6 c.886+2_886+5del r.(spl?) EGF2 maternal L, H, E?, V?, F This study
34 IVS 10 c.1348+1G>A r.spl? EGF6 ND L, E, F This study
35 IVS 11 c.1395+3A>G r.(spl?) EGF7 de novo L, H, E, F Jurkiewicz et al. (2006)
36 IVS 24 c.3048+1_3048+2insG r.(spl?) CR-TM de novo L, H, E, V, F, R Jurkiewicz et al. (2005)
37 IVS 24 c.3048+5_3048+7delGTA r.(spl?) CR-TM maternal L, H, E, V, F, R Röpke et al. (2003)
Missense 38
e
Ex 2 c.359T>A p.(Ile120Asn) SP-DSL maternal L, H, E, V, F Jurkiewicz et al. (2005)
39
e
Ex 2 c.359T>A p.(Ile120Asn) SP-DSL maternal L, H, E, F Jurkiewicz et al. (2005)
40 Ex 4 c.551G>A p.(Arg184His) SP-DSL paternal L, E, F, R Giannakudis et al. (2001)
41 Ex 4 c.551G>A p.(Arg184His) SP-DSL paternal L, H, E, V, F Jurkiewicz et al. (2006)
42 Ex 4 c.560G>A p.(CysC187Tyr) DSL NM L, E, V, F Jurkiewicz et al. (2005)
43 Ex 4 c.672G>T p.(Trp224Cys) DSL de novo L, H, F Röpke et al. (2003)
44 Ex 9 c.1156G>A p.(Gly386Arg) EGF5 ND L, H, V, F This study
45 Ex 9 c.1156G>A p.(Gly386Arg) EGF5 de novo L, H, E, F This study
46 Ex 10 c.1286A>C p.(Asn429Thr) EGF6 maternal L, H, E, F This study
47 Ex 10 c.1312T>G p.(Cys438Gly) EGF6 maternal L, H, E, F This study
Large genomic
rearrangements
48 Ex 20–23 c.2889-?_3376+?del EGF15 – CR de novo L, H, E, V, F, R This study
g
49 Ex 3–25 c.916-?_3999+?del SP-DSL –CR-TM ND L, H, E, V, F, R This study
g
50 Ex 1–25 53.9 kb deletion, breakpoints:
10,570,644-10,624,536
gene deletion SP – CR-TM de novo L, H, E, F, R This study
g
51 Whole gene gene deletion all de novo L, H, E, F This study
J Appl Genetics (2014) 55:329–336 331

mutations were identified including seven frameshift, five
nonsense, three missense, two splice-site, and six gross
deletions. Thirteen novel point mutations were detected.
All of the identified JAG1 point mutations map into the
extracellular domain of the JAG1 protein and are distribut-
ed throughout the JAG1 gene. Seventy-two percent (13/18)
of all point mutations are localized in epidermal growth
factor (EGF) repeat regions and 66 percent of them (seven
frameshift and five nonsense) are expected to lead to pre-
mature termination codons. All missense mut ations not
described previously (p.Asn429Thr, p.Cys438Gly) were
predicted to be probably damaging to the protein function in
the in silico analyses performed by means of both PolyPhen2
and SIFT software.
In one patient a substitution c.2048G>A (p.Arg683His)
was identified but predictions from PolyPhen2 and SIFT
described it as a benign change, thus it was considered a rare
polymorphism. The change was not repo rted be fore.
Molecular analysis of mother’s DNA did not reveal the
change and the father’s DNA was not available.
The MLPA analysis revealed partial JAG1 deletions in two
patients (deletion of exons 3–25 and deletion of exons 20–23)
and whole JAG1 gene deletions in six patients (including two
siblings and their paternal aunt). The exact size of the genomic
alterations was further evaluated by aCGH. In one familial
case of ALGS all three affected members carried the same
deletion on chromosome 20, which was predicted to span at
least 5.4 Mb. The deletions for patients no. 51 and 52 are
predicted to span at least 991 kb and 2.26 Mb, respectively.
Sequencing of the NOTCH2 gene in five patients in whom
we did not identify a JAG1 mutations or large deletion did not
reveal any pathogenic alterations. We were unable to screen
the NOTCH2 gene in four JAG1 negative patients.
Discussion
Mutational analysis of the coding sequence of the JAG1 gene
in a cohort of 35 Polish ALGS patients has revealed 26
patients with mutations. Combined with the Polish ALGS
patients previously reported in the literature (30 patients) we
identify a cohort of JAG1 positive Polish ALGS patients, with
mutations in 56 patients coming from 53 families
(Giannakudis et al. 2001; Stankiewicz et al. 2001; Röpke
et al. 2003; Jurkiewicz et al. 2005, 2006) (Table 1). Fifty
different mutations were found. All of the identified mutations
are localized in the extracellular domain of the JAG1 protein.
Fifty-six percent of various point mutations map into epider-
mal growth factor (EGF) repeat regions. Sixty-five percent of
point mutations (frameshift and nonsense mutations) are pre-
dicted to lead to premature termination codons. Most of the
mutations were private, only three various mutations (c.439+
1G>A, c.551G>A, c.1156G>A) were recurrent and each of
them occurred twice in unrelated patients.
Table 1 (continued)
Patient
No.
Exon or intron Mutation position
a
Predicted
consequence
Protein domain
b
Origin
c
Phenotype
d
Reference
991 kb deletion, breakpoints:
9,818,619–10,810,007
52 Whole gene 2.26 Mb deletion, breakpoints:
9,272,721–11,534,825
gene deletion all de novo L, H, F, R This study
53
e
Whole gene 5.4 Mb deletion, breakpoints:
9,323,011–14,733,354
gene deletion all paternal L, H, E, V, F, R This study
g
54
e
Whole gene 5.4 Mb deletion, breakpoints:
9,323,011–14,733,354
gene deletion all paternal L, H, E, V, F This study
g
55
f
Whole gene 5.4 Mb deletion, breakpoints:
9,323,011–14,733,354
gene deletion all ND L, H, E, V, F, R This study
g
56 Whole gene paracentric inversion of
chromosome 20p12.2p13,
insertion breakpoint
between exons 5 and 6
of JAG1 gene
EGF1 de novo L, H, E, F Stankiewicz et al. (2001)
a
the JAG1 sequence is that of the cDNA of the GenBank accession no. NM_000214.2; the nucleotide position at the A of the ATG translation start codon
is denoted as nucleotide +1; novel point mutations appear in boldface print; chromosomal coordinates are given according to the GRCh37/hg19
assembly
b
SP-DSL – region between signal peptide and DSL domain, DSL – Delta/Serrate/Lag2 domain, EGF – epidermal growth factor repeats domain, CR –
cysteine-rich region, CM-TM – region between CR and TM, TM – transmembrane domain
c
ND – not determined, parent’s samples not available; NM – mutation not detected in mother’ssample,father’s sample not available
d
main ALGS symptoms: L – liver, H – heart, E – eye, V – vertebrae, F – face, R – renal involvement, ? – the feature was not examined
e
siblings
f
cousin of the siblings no. 53, 54
g
patients from previous studies eveluated again in this study
332 J Appl Genetics (2014) 55:329–336

Over 440 various JAG1 mutations have already been de-
scribed in ALGS patients. The most common are frameshift
mutations (49 %), followed by nonsense mutations (16 %),
missense mutations (15 %), gross deletions and insertions
(11 %), while the least frequent variants are splice site muta-
tions (9 %) (Li et al. 1997; Oda et al. 1997;Krantzetal.1998;
Yuan et al. 1998, 2001; Crosnier et al. 1999, 2000;Onouchi
et al. 1999; Pilia et al. 1999; Heritage et al. 2000, 2002;
Colliton et al. 2001; Giannakudis et al. 2001; Röpke et al.
2003;Jurkiewiczetal.2005; Warthen et al. 2006;Kamath
et al. 2009;Gueganetal.2012; Lin et al. 2012;Wangetal.
2012). The spectrum of JAG1 mutations in the Polish ALGS
patients presents a similar pattern to those from other groups,
with only slight differences in the frequency of some types of
mutations. As in previously reported studies, frameshift muta-
tions were the most frequent (34 %), however , nonsense mu-
tations and splice s ite mutations were more common than in
other populations (22 % and 16 %, respectively). The frequen-
cy of missense mutations and large genomic rea rrangements
was almost the same as in other studies (14 % in both groups).
Mutations identified in the Polish cohort were spread over
almost the entire JAG1 gene except exons 1, 8, 19, 20, and 21
(Fig. 1). Forty-two percent of point mutations are found in
four exons (2, 4, 9, 10) and 81 percent of point mutations are
found in 12 exons (2, 3, 4, 5, 6, 9, 10, 12, 14, 18, 22, 24). No
hot spot was found. In other reported populations JAG1 mu-
tations are also distributed along the whole gene, with 47
percent of point mutations localized in six exons (2, 4, 6, 17,
23, 24). Exons 2 and 4 are fragments of the JAG1 gene with
the highest number of mutations both in the Polish cohort and
other populations.
Mutation screening of the JAG1 gene in Polish patients in
previous studies was mainly performed by SSCP followed by
sequencing of selected fragments (Jurkiewicz et al. 2005,
2006). To see how the limitations of the SSCP technique
decreases the mutation detection rate in this large gene, 13
patients from 11 unrelated families in whom mutations were
not revealed were subject to JAG1 sequencing. The MLPA
analysis was also performed in this group of patients as it was
not implemented in the previous studies. Only patients with
classic presentation of ALGS were included in the mutation
re-evaluation. The sequencing revealed nonsense mutations
(c.703C>T, c.1325G>A) in two unrelated patients, consistent
with single nucleotide substitutions having a higher likelihood
of being missed by SSCP analysis. Moreover, MLPA screening
has revealed gross deletions in six patients from four families
that further underlines the usefulness and importance of that
technique in ALGS diagnostics (Kamath et al. 2009;Linetal.
2012). In five patients negative for
JAG1 mutations the
NOTCH2 gene sequencing revealed no pathogenic changes.
Fig. 1 Distribution of JAG1
mutations in ALGS patients. a:
Polish cohort. b: Other reported
populations
J Appl Genetics (2014) 55:329–336 333

Overall, JAG1 mutations were found in 53 Polish ALGS
patients out of a group of 62 unrelated patients, and therefore,
the mutation detection rate in the Polish cohort is 85 percent.
When patients from both this study and the literature are
considered, analysis of parental samples was conducted in 43
families and revealed that mutations were inherited in 37 % of
cases. In two families the presence of parental mosaicism was
proved (Giannakudis et al. 2001), which should be taken into
account in the diagnosis and genetic counseling. Analysis of
parents for whom clinical data were available revealed that
most of them were unaffected or presented only mild ALGS
features such as the characteristic facial features or posterior
embryotoxon. Three mothers had heart defects and two
mothers had both heart and liver manifestations. Such a di-
verse clinical manifestation in individuals carrying the same
primary disease causing mutation suggests a role for genetic
modifiers of the clinical outcome.
The large size of the JAG1 gene makes diagnostics of ALGS
labor intensive. However , analysis of the frequency and distri-
bution of mutations along the JAG1 gene enables us to propose
an effective diagnostic strategy for Polish ALGS patients
(Fig. 2). We suggest a tiered approach, with initial sequencing
of the exons with the largest number of mutations (four or
more), followed by a second tier (2–3 per exon), followed by
sequencing of exons in which one or no mutation has been
found so far. In patients negative for JAG1 point mutations,
MLPA screening for large deletions involving JAG1 gene
should be executed. The JAG1 gene analysis might be com-
pleted by the NOTC H2 gene screening, however the analysis of
this gene seems to be of minor importance in the ALGS routine
diagnostics as NOTCH2 mutations have been reported so far
only in ten patients with ALGS features (McDaniell et al. 2006;
Kamath et al. 2012). The proposed strategy is b ased on
methods commonly available in most laboratories, primarily
Sanger sequencing. However , new molecular technologies are
evolving rapidly fueled by the utilization of next generation
sequencing (NGS) based tests (Pareek et al. 2011; Rabbani
et al. 2014). This technology allows the simultaneous analysis
of selected portions of the genome, such as several or many
genes, or the whole exome or the whole genome. As new
methods become more cost ef fective and accessible, the pro-
posed diagnostic strategy will have to be updated.
Suspected diagnosis of ALGS
Screening for point mutations in exons 2, 4, 9, 10 of JAG1 gene
MLPA screening for large deletions
of JAG1 gene
Screening for point mutations in remaining
fourteen exons of JAG1 gene
Screening for point mutations in exons
3, 5, 6, 12, 14, 18, 22, 24 of JAG1 gene
Diagnosis confirmed
Genetic testing of parents/relatives
Eventual screening for mutations in
NOTCH2 gene
-ve
+
ve
-ve
+
ve
-ve
+
ve
+
ve -ve
Consider an alternative diagnosis
-ve
+
ve
Fig. 2 Diagram of proposed
genetic investigations for
suspected Polish ALGS patients
334 J Appl Genetics (2014) 55:329–336

In accordance with previous reports, no apparent correla-
tion between genotype and phenotype was observed. In com-
paring the clinical phenotype of the Polish cohort with JAG1
mutations to other cohorts, the liver phenotype occurs in
100 % and cardiac phenotype occurs in 89 % of the population
(Table 1), whereas both these features are present in over 94 %
of other reported populations (Warthen et al. 2006; Lin et al.
2012). Renal involvement is observed in 21 % of Polish
ALGS patients with JAG1 mutations, while it occurs in 27–
39 % of ALGS patients in other cohorts (Warthen et al. 2006;
Kamath et al. 2001; Lin et al. 2012).
This study presents a comprehensive analysis of JAG1
mutations in the cohort of Polish patients with ALGS. The
review of all patients from the current and previous studies
allows us to determine the genetic background of this popu-
lation and to propose an effective diagnostic strategy for
Polish ALGS patients.
Acknowledgments We thank the families and the physicians who have
submitted samples for these studies. The study was supported in part by
the Children’s Memorial Health Institute in Warsaw, statutory project no.
212/10 and EU Structural Funds, project POIG.02.01.00-14-059/09. The
authors declare no conflict of interest.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.
References
Alagille D, Estrada A, Hadchouel M, Gautier M, Odievre M,
Dommergues JP (1987) Syndromic paucity of interlobular bile ducts
(Alagille syndrome or arteriohepatic dysplasia): review of 80 cases.
J Pediatr 110:195–200
Colliton RP, Bason L, Lu F-M, Piccoli DA, Krantz ID, Spinner NB
(2001) Mutation analysis of Jagged1 (JAG1) in Alagille syndrome
patients. Mutation in Brief #397. Online. Hum Mutat 17:151–152
Crosnier C, Driancourt C, Raynaud N, Dhorne-Pollet S, Pollet N, Bernard
O, Hadchouel M, Meunier-Rotival M (1999) Analysis of mutations
of the Jagged1 gene in patients with Alagille syndrome: evidence for
most cases being sporadic. Gastroenterology 116:1141–1148
Crosnier C, Driancourt C, Raynaud N, Hadchouel M, Meunier-Rotival M
(2000) Novel mutations in the JAGGED1 gene of patients with Alagille
syndrome. Mutation in Brief #385 (Online). Hum Mutat 17:72–73
Dhorne-Pollet S, Deleuze JF, Hanchouel M, Bonaiti-Pellie C (1994)
Segregation analysis of Alagille syndrome. J Med Genet 31(6):453–457
Emerick KM, Rand EB, Goldmutz E, Krantz ID, Spinner NB, Piccoli DA
(1999) Features of Alagille syndrome in 92 patients: frequency and
relation to prognosis. Hepatology 29:822–829
Giannakudis J, Röpke A, Kujat A, Krajewska-Walasek M, Hughes H,
Fryns JP, Bankier A, Amor D, Schlicker M, Hansmann I (2001)
Parental mosaicism of JAG1 mutations in families with Alagille
syndrome. Eur J Hum Genet 9:209–216
Guegan K, Stals K, Day M, Turnpenny P, Ellard S (2012) JAG1 muta-
tions are found in approximately one third of patients presenting
with only one or two clinical features of Alagille syndrome. Clin
Genet 82:33–40
Heritage M, MacMillan JC, Colliton RP, Genin A, Spinner NB, Anderson
GJ (2000) Jagged1 (JAG1) mutation detection in an Australian
Alagille syndrome population. Hum Mutat 16:408–416
Heritage ML, MacMillan JC, Anderson GJ (2002) DHPLC mutation
analysis of Jagged1 (JAG1) reveals six novel mutations in
Australian Alagille syndrome patients. Hum Mutat 20(6):481
Jurkiewicz D, Popowska E, Glaser C, Hansmann I, Krajewska-Walasek M
(2005) T welve novel JAG1 gene mutations in Polish Alagille syn-
drome patients. Mutation in Brief #784. Online. Hum Mutat 25:321
Jurkiewicz D, Popowska E, Krajewska-Walasek M (2006) Udział genu
JAG1 w patogenezie zespołuAlagille’a u polskich pacjentów.
Standardy Medyczne T.3 nr 5:490–496
Kamath BM, Podkameni G, Hutchinson AL, Leonard LD, Gerfen J,
Kranz ID, Picccoli DA, Spinner NB, Loomes KM, Meyers K
(2001) Renal anomalies in Alagille syndrome: a disease-defining
feature. Am J Med Genet A 158A:85–89
Kamath BM, Thiel BD, Gai X, Conlin LK, Munoz PS, Glessner J, Clark
D, Warthe DM, Shaikh TH, Mihci E, Piccoli DA, Grant SF,
Hakonarson H, Krantz ID, Spinner NB (2009) SNP array mapping
of chromosome 20p deletions: Genotypes, phenotypes, and copy
number variation. Hum Mutat 30:371–378
Kamath BM, Bauer RC, Loomes KM, Chao G, Gerfen J, Hutchinson A,
Hardikar W, Hirshfield G, Jara P, Krantz ID, Lapunzina P, Leonard L,
Ling S, Ng VL, Hoang PL, Piccoli DA, Spinner NB (2012) NOTCH2
mutations in Alagille syndrome. J Med Genet 49:138–144
Krantz ID, Colliton RP, Genin A, Rand EB, Li L, Piccoli DA, Spinner NB
(1998) Spectrum and frequency of Jagged1 (JAG1) mutations in
Alagille syndrome patients and their families. Am J Hum Genet 62:
361–1369
Li L, Krantz ID, Den Y, Genin A, Banta AB, Collins CC, Qi M, Trask BJ, Wl
K, Cochran J, Costa T, Pierpont MEM, Rand EB, Piccoli DA, Hood L,
Spinner NB (1997) Alagille syndrome is caused by mutations in human
Jagged1, which encodes a ligand for Notch1. Nat Genet 16:243–251
Lin HC, Hoang PL, Hutchinson A, Chao G, Gerfen J, Loomes KM,
Krantz I, Kamath BM, Spinner NB (2012) Alagille syndrome in a
Vietnamese cohort: mutation analysis and assessment of facial fea-
tures. Am J Med Genet A 158A:1005–1013
Lindsell CE, Shawber DJ, Boulter J, Weinmaster G (1995) Jagged: a
mammalian ligand that activates Notch1. Cell 80:909–917
McDaniell R, Warthen DM, Sanchez-Lara PA, Pai K, Kranz ID, Piccoli
DA, Spinner NB (2006) NOTCH2 mutations cause Alagille syn-
drome, a heterogeneous disorder of the Notch signaling pathway.
Am J Hum Genet 79:169
–171
Oda T, Elkahloun AG, Pike BL, Okajima K, Krantz ID, Genin A, Piccoli
DA, Meltzer PS, Spinner NB, Collins FS, Chandrasekharappa SC
(1997) Mutations in the human Jagged1 gene are responsible for
Alagille syndrome. Nat Genet 16:235–242
Onouchi Y, Kurahashi H, Tajiri H, Ida S, Okada S, Nakamura Y (1999)
Genetic alternations in the JAG1 gene in Japanese patients with
Alagille syndrome. J Hum Genet 44:235–239
Pareek CS, Smoczynski R, Tretyn A (2011) Sequencing technologies and
genome sequencing. J Appl Genet 52:413–435
Pilia G, Uda M, Macis D, Frau F, Crisponi L, Balli F, Barbera C,
Colombo C, Frediani T, Gatti R, Iorio R, Marazzi MG, Marcellini
M, Musumeci S, Nebbia G, Vajro P, Ruffa G, Zancan L, Cao A, De
Virgilis S (1999) Jagged-1 mutation analysis in Italian Alagille
syndrome patients. Hum Mutat 14:394–400
Rabbani B, Tekin M, Mahdieh N (2014) The promise of whole-exome
sequencing in medical genetics. J Hum Genet 59:5–15
Röpke A, Kujat A, Graber M, Giannakudis J, Hansmann I (2003)
Identification of 36 novel Jagged 1 (JAG1) mutations in patients
with Alagille syndrome. Hum Mutat 21(1):100
Stankiewicz P, Rujner J, Lof fler C, Kruger A, Nimmakayalu, Piłacik B,
Krajewska-Walasek M, Gutkowska A, Hansmann I, Giannakudis I
(2001) Alagille syndrome associated with a paracentric inversion
20p12.2p13 disrupting the JAG1 gene. Am J Med Genet 103:166–171
J Appl Genetics (2014) 55:329–336 335

Wang H, Wang X, Li Q, Chen S, Liu L, Wei Z, Wang L, Liu Y, Zhao X,
He L, Wang J, Xing Q (2012) Analysis of JAG1 gene variant in
Chinese patients with Alagille syndrome. Gene 499:191–193
Warthen DM, Moore EC, Kamath BM, Morrissette JJD, Sanchez P,
Piccoli DA, Krantz ID, Spinner NB (2006) Jagged1 (JAG1) muta-
tions in Alagille syndrome: Increasing the mutation detection rate.
Hum Mutat 27:436–443
Yuan ZR, Zohsaka T, Ikegaya T, Suzuki T, Okano S, Abe J, Kobayashi N,
Yamade M (1998) Mutational analysis of the Jagged1 gene in
Alagille syndrome families. Hum Mol Genet 7:1363–1369
Yuan ZR, Okaniwa M, Nagata I, Tazawa Y, Ito M, Kawarazaki H,
Inomata Y, Okano T, Yoshida T, Kobayashi N, Kohsaka T (2001)
The DSL domain in mutant JAG1 ligand is essential for the severity
of the liver defect in Alagille syndrome. Clin Genet 59:330–337
336 J Appl Genetics (2014) 55:329–336