Isolated aneurysmal disease as an underestimated finding in
individuals with JAG1 pathogenic variants.
Jotte Rodrigues Bento1, Alice
Krebsová2, Ilse Van Gucht1, Irene
Valdivia Callejon1, An Van
Berendoncks3, Pavel Votypka4, Ilse
Luyckx1,5, Petra Peldova4, Steven
Laga6, Marek Havelka4, Lut Van
Laer1, Pavel Trunecka7, Nele
Boeckx1, Aline Verstraeten1, Milan
Macek4, Josephina A.N. Meester1,
Bart Loeys1,5
1Centre of Medical Genetics, Antwerp University
Hospital/University of Antwerp, Antwerp, Belgium
2Department of Cardiology, Center for Inherited
Cardiovascular Disorders, Institute for Clinical and Experimental
Medicine (IKEM), Praha 4, 14021 Prague, Czech Republic
3Department of Cardiology, Antwerp University
Hospital/University of Antwerp, Antwerp, Belgium
4Department of Biology and Medical Genetics, Second
Faculty of Medicine, Charles University and Motol University Hospital,
Praha 5, 15006 Prague, Czech Republic
5Department of Human Genetics, Radboud University
Medical Center, Nijmegen, The Netherlands
6Department of cardiac surgery, Antwerp University
Hospital/University of Antwerp, Antwerp, Belgium
7Department of Hepatology and Gastroenterology,
Transplant Center of Institute for Clinical and Experimental Medicine
(IKEM), Praha 4, 14021 Prague, Czech Republic
Abstract: Pathogenic variants in JAG1 are known to cause
Alagille syndrome (ALGS), a disorder that primarily affects the liver,
lung, kidney and skeleton. Whereas cardiac symptoms are also frequently
observed in ALGS, thoracic aortic aneurysms have only been reported
sporadically in post-mortem autopsies. We here report two families with
segregating JAG1 variants that present with isolated aneurysmal
disease, as well as the first histological evaluation of aortic aneurysm
tissue of a JAG1 variant carrier. Our observations shed more
light on the pathomechanisms behind aneurysm formation in JAG1variant carriers and underline the importance of cardiovascular imaging
in the clinical follow-up of JAG1 variant carrying individuals.
Keywords: Alagille syndrome, JAG1 , thoracic aortic
aneurysm, intracranial aneurysm
Main text: JAG1 serves as one of five Notch interacting
surface ligands and is ubiquitously expressed in the embryo, while
expression in adult tissue is restricted to the heart, placenta,
pancreas, prostate and large arteries (Carithers & Moore, 2015;
Grochowski, Loomes, & Spinner, 2016). Given that the Notch pathway is a
highly conserved signaling cascade engaged in normal development of
fetal organs and in cell fate decisions in postnatal life, it is not
unexpected that variants in Notch pathway components can cause various
diseases (Grochowski et al., 2016). Alagille syndrome (ALGS) is one such
rare heritable disorder that is predominantly caused by loss-of-function
variants in JAG1 (in 94.3% of ALGS patients, another 2.5% hasNOTCH2 variants, 3.2% of patients remains) and has an estimated
incidence of 1:30,000 live births (Gilbert et al., 2019). ALGS is
clinically diagnosed when three out of seven typical clinical features
are observed, but due to marked variability (even within families),
patients can remain undiagnosed. A molecular diagnosis is therefore the
key to adequate patient management (Saleh, Kamath, & Chitayat, 2016).
Hepatic abnormalities, with
intrahepatic duct deficiency leading to cholestasis as the most
prevalent manifestation, are observed in up to 100% of patients;
posterior embryotoxon has a prevalence of approximately 80-90%; and
cardiac structural changes such as Tetralogy of Fallot and peripheral
pulmonary artery stenosis are present in over 90% of patients (Saleh et
al., 2016; Spinner, Gilbert, Loomes, & Krantz, 1993). Renal disease,
distinct facial features, and butterfly and hemi-vertebrae are also
reported frequently. Furthermore, non-cardiac vascular complications
were noted to be a significant part of the clinical spectrum (estimated
in 34% of ALGS patients), with intracranial aneurysm and hemorrhage as
the most frequent finding (Kamath et al., 2004; Saleh et al., 2016;
Spinner et al., 1993).
Using whole exome sequencing on the DNA of a fetus presenting with left
hypoplastic heart syndrome and left renal agenesis (Figure 1A IV:4), a
missense variant in JAG1 (NM_000214:c.2242T>C,
p.Cys748Arg, ClinVar accession VCV001172525.1) was discovered. The
variant (i) replaces a highly conserved and critical cysteine residue
that is part of a disulfide bridge in one of the 14 EGF repeats, which
are important for Notch receptor binding; (ii) is absent from control
databases (including GnomAD); (iii) has not been previously reported;
and (iv) was the only relevant pathogenic variant identified in this
patient (Ashkenazy et al., 2016; Grochowski et al., 2016; Karczewski et
al., 2020). The JAG1 variant found in the fetus was inherited
from the mother (Figure 1A III:6, proband, 35 years old), who underwent
surgery at the age of 10 for aortic coarctation, a feature that has been
linked to ALGS before (Kamath et al., 2004). Examination of the mother
and her siblings revealed absence of typical ALGS manifestations.
However, in both the monozygotic twin sister (Figure 1A III:8) and
brother (36 years old, Figure 1A III:11), thoracic aortic aneurysms
(TAA) were discovered with a diameter of 40 mm (Z-score of 3.6) and 58
mm (Z-score of 8.6) respectively, urging the brother to undergo
immediate Bentall surgery with mechanical aortic valve replacement.
Histological examination of aorta obtained during surgery showed
fragmentation of elastic fibers with marked decrease in elastin content
(Figure 2A and 2B). Collagen was drastically increased and disorganized
compared to a healthy control sample (Figure 2C and 2D). Since the
histological phenotype in our patient resembles that in Marfan syndrome
and Loeys-Dietz syndrome patients (Maleszewski, Miller, Lu, Dietz, &
Halushka, 2009) and dysregulated TGFβ signaling is a key feature of
these syndromes, we also studied whether phosphorylated SMAD2 (pSMAD2),
a downstream marker of TGFβ activity, is increased here too. Indeed, we
qualitatively observed that more nuclei were positive for pSMAD2 (Figure
2F) compared to a control sample (Figure 2E). The father of the proband
(Figure 1A II:7, aged 69) carried the variant and did not show any
features besides aortic wall calcifications. Segregation analysis
revealed presence of the variant in three siblings (II:2, II:5 and II:6;
Figure 1A). Of note, the uncle of the proband (II:2 63 years old)
suffered from multiple intracranial aneurysms (IA) and subarachnoid
bleeding from ruptured arteria communicans anterior aneurysm. The aunt
(II:5, aged 65) presented with bicuspid aortic valve but normal aortic
measurements (sinus 34 mm, aorta ascendens 34 mm). The second aunt
(II:6; aged 60) had normal echocardiographic evaluation. This marked
inter-familial phenotypic variability reflects the significant and
repeatedly described range in disease expression of ALGS (Gilbert et
al., 2019; Guegan, Stals, Day, Turnpenny, & Ellard, 2012). Individual
IV:6 had a normal echocardiographic evaluation, but she is only 3 years
old. Intriguingly, apart from the cardiac and renal phenotype of the
fetus (Figure 1A IV:4), none of the patients showed any of the typical
ALGS features.
In a second family, sequencing of a custom-made cardiac/aortic
conditions-related gene panel revealed deletion-insertion variant ofJAG1 in the 42-year-old male TAA proband (Figure 1B, II:1). The
variant affects the splice acceptor site and first nucleotides of exon
17(NM_000214.2: c.2114-5_2119delins18). The variant was not reported
before and RNA analysis of the proband revealed in-frame skipping of
exon 17 (Figure S1). No other relevant genetic variants were identified.
At age 24 years, the patient underwent Bentall surgery with mechanical
aortic valve replacement because of an accidental finding of aortic
aneurysm (at age 22, ascending aorta 60 mm, Z-score of 10). Other family
members, including the proband’s sister who also carried the
deletion-insertion variant, also presented with severe vascular disease.
The mother of the proband (Figure 1B, I:2) underwent preventive surgery
with aortic replacement for TAA at the age of 61 and died at the age of
63 of an intracranial bleeding associated with IA. The proband’s sister
(Figure 1B, II:2) suffered multiple IAs (left basilary and cerebral
artery, arteria cerebri posterior, bilateral arteria cerebri media) and
subarachnoid bleeding events, but showed normal aortic dimensions. The
proband’s daughter (Figure 1B, III:2 - age 13 years), which carries the
mutation, had a normal total body MR angiography. In accordance with the
first family, patients in family B (Figure 1B, II:2, II:1, II:2) did not
present with typical ALGS hepatic, ocular or skeletal characteristics.
We are the first to report two ALGS families with isolated aneurysmal
disease lacking other ALGS pathognomonic clinical characteristics (such
as the hepatic abnormalities, posterior embryotoxon and cardiac
structural changes described above). In the current ALGS literature
there is no notion of TAA in the vast majority of ALGS patient
descriptions, but systematic aortic evaluation might not be performed on
a routine basis. TAA has been reported before in only four ALGS
patients, three of which were discovered at autopsy (Kamath et al.,
2004; Molinero-Herguedas, Labrador-Fuster, Rios-Lazaro, &
Carmaniu-Tobal, 2008). In all four, at least one typical clinical ALGS
manifestation was present. In the patient reported by Molinero-Herguedas
et al., TAA was discovered during reexamination of an (undefined)
cardiopathy and three patients reported in Kamath et al. had
characteristic facial features, but due to discovery at autopsy no
details on other organ system involvement were available. Similar for IA
in ALGS, which has an estimated prevalence of 14%, patients previously
reported always presented accompanying cholestatic liver disease and
cardiac defects (Kamath et al., 2004).
For the first time, histological evaluation of aortic tissue of a TAA
patient with a JAG1 variant was performed, revealing an
unexpected phenotype with elastin degradation and abnormal collagen
deposition. Fibrosis has been associated with JAG1gain-of-function rather than loss-of-function in fibrotic kidney
disease, where excessive Notch signaling is known to induce
disproportionate expression of extracellular matrix (ECM) components (Hu
et al., 2015). Additionally, in a mouse model of abdominal aortic
aneurysm, pharmacological inhibition of Notch signaling resulted in
regression of aneurysm along with a reduction in elastic fiber
fragmentation and collagen deposition (Sharma et al., 2019). On the
other hand, it has been observed that dysfunction of Notch signaling
mitigates epithelial-to-mesenchymal transition (EMT), an indispensable
process during cardiovascular development and repair mechanisms.
Additionally, contractile markers of vascular smooth muscle cells (VSMC)
are downregulated upon Notch impairment, suggesting that the VSMC in the
aortic media of ALGS patients adopt a synthetic phenotype, characterized
by increased ECM synthesis and elastolysis due to secretion of matrix
metalloproteases. Resulting tissue damage and subsequent failure to
sufficiently dampen hemodynamical pressure could trigger TGFβ signaling,
which is expressed by increased pSMAD2 in our patient. Knowing that TGFβ
is a well-known mediator of fibrosis and a driver of metalloprotease
activity, a destructive cycle is initiated, leading to aortic wall
degradation (Jones, Spinale, & Ikonomidis, 2009; Kostina et al., 2016;
Zavadil, Cermak, Soto-Nieves, & Bottinger, 2004). Further functional
studies will be necessary to determine the true sequence of events of
ALGS related TAAD.
With the current report of these patients with significant Z-scores and
intracranial hemorrhaging at a relatively young age without obvious ALGS
findings, we want to urge clinicians to include JAG1 (andNOTCH2 ) in genetic screening panels for TAA and IA as this is not
yet the case in most clinics – most probably due to insufficient
clinical evidence (Renard et al., 2018), which we now provide.
Furthermore, we anticipate TAA might be an underestimated finding in
ALGS and we therefore advise to early and thoroughly examine the
vascular system in such patients, with focus on intracranial and
thoracic aorta imaging, since vascular events are estimated to account
for over 30% of ALGS mortality (Kamath et al., 2004). A systematic
large-scale ALGS cohort investigation could straighten out the true
significance of aortopathy in ALGS.