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\begin{document}

\title{NT5E mutation in sisters who underwent aortic valve replacements for
aortic stenosis}



\author[1]{Tetsuro Uchida}%
\author[2]{Atsushi Yamashita}%
\author[2]{Ai Ishizawa}%
\author[3]{Mitsuaki Sadahiro}%
\author[4]{Nobuyoshi Azuma}%
\author[5]{Tadashi Kaname}%
\affil[1]{Yamagata University}%
\affil[2]{Yamagata University Faculty of Medicine}%
\affil[3]{Yamagata Daigaku Igakubu}%
\affil[4]{Asahikawa Medical University}%
\affil[5]{National Center for Child Health and Development}%


\vspace{-1em}



  
  \date{May 27, 2024}


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\selectlanguage{english}
\begin{abstract}
Background and Aims: Mutations of the NT5E gene encoding the cluster of
differentiation 73 (CD73) protein have been found in patients with
characteristic calcification of joints and arteries (CALJA). CD73 plays
a protective role against aortic valve calcification and its deletion
results in aortic valve calcification. However, there have been no
reports of a patient with CALJA with aortic stenosis. Methods: We
describe two extremely rare cases of two sisters with identical NT5E
gene mutation patterns, both of whom presented severe aortic stenosis
and limb ischemia. Genetic examination for definitive diagnosis was also
performed in both patients. Results: Both patients underwent aortic
valve replacement and bilateral distal arterial bypass surgeries
successfully. They were genetically diagnosed with CALJA based on the
NT5E mutation. Conclusions: NT5E mutation should be considered in
patients requiring aortic valve replacement for a calcified aortic valve
and bypass surgery for specific calcified and occluded arteries.%
\end{abstract}%



\sloppy


\textbf{Introduction}

In 2011, mutation of the NT5E gene, that encodes cluster of
differentiation (CD) 73, was found to result in specific calcifications
in patients with aneurysmal dilatation of arteries as well as joint
calcifications. These symptoms were termed calcification of joints and
arteries (CALJA).\textsuperscript{1} Since then, only 14 patients from 7
families have been reported as having
CALJA.\textsuperscript{2-5}Patients with CALJA often experience ischemic
symptoms in the lower extremities secondary to severely calcified and
occluded arteries. CD73 is also known to play a protective role against
aortic valve calcification by hydrolyzation of adenosine monophosphate
(AMP) to adenosine.\textsuperscript{6} Although the deletion of the CD73
gene has been reported to cause aortic valve calcification leading to
valvular stenosis in animal models,\textsuperscript{7} there are no
reports of patients with CALJA requiring an aortic valve replacement
(AVR) for aortic stenosis (AS). We present two extremely rare cases of
sisters with identical NT5E gene mutation patterns, both of whom
underwent AVR for AS.

\textbf{Materials and Methods}

Both sisters underwent AVR for AS and distal bypass surgeries for
chronic limb-threatening ischemia. As they presented with the typical
features of CALJA, both patients underwent a genetic examination for
definitive diagnosis of CALJA.

Genomic DNA was extracted from whole blood cells using a QIAamp DNA
Blood Mini Kit (QIAGEN, Hilden, Germany). We performed whole exome
sequencing analysis as previously described.\textsuperscript{8} In
brief, we used the SureSelect Human All Exon V6 kit (Agilent Technology,
Santa Clara, CA) for capture and a HiSeq2500 (Illumina, San Diego, CA)
for sequencing. Reads were aligned to GRC37 using Burrows-Wheeler
Aligner (http://bio-bwa.sourceforge.net/). Variants were identified
using the GATK Unified Genotyper and ANNOVA (http://
annover.openbioinfomatics.org/en/latest/).

This genetic analysis of the patients was approved by the Ethics
Committee of Yamagata University. Both patients and their family members
provided informed consent for clinical and genetic examinations and for
the publication of this report.

\textbf{Results}

\textbf{Patient 1} : An 81-year-old woman presented with a 50-year
history of joint pain in her legs. She underwent bilateral distal bypass
surgeries at 72 years of age for intermittent claudication. Plain
radiography of the lower extremities revealed significant calcification
with areas of arteriomegaly. Three-dimensional computed tomography
revealed extensive occlusion of the bilateral femoropopliteal arteries
with aneurysmal changes and severe calcifications, which are typical
findings of CALJA (Fig. 1A). The patient's saphenous vein grafts were
patent when she presented to us. During her recovery from the vascular
reconstructive surgeries, the patient gradually developed exertional
fatigue. Echocardiography revealed severe aortic valve stenosis with a
peak pressure gradient of 50 mmHg. AVR was performed when the patient
was 77 years old. The native aortic valve was tricuspid and the valvular
leaflets and annulus showed significant calcification (Fig. 1B).
However, no calcification was observed in the aortic wall. She underwent
AVR with a Carpentier-Edwards PERIMOUNT bioprosthetic valve (Edwards
Lifesciences, Irvine, CA) (Fig. 1C). Her postoperative course was
uneventful.

\textbf{Patient 2} : A 74-year-old woman, who is the younger sister of
Patient 1, reported a 10-year history of bilateral intermittent
claudication of the calves and chronic coldness of her lower
extremities. At 71 years of age, she had developed dyspnea on exertion
and was diagnosed with severe aortic valve stenosis with a peak pressure
gradient of 90 mmHg. Her native aortic valve was tricuspid with severe
calcification of the cusps and annulus (Fig. 2A). There were no
calcified lesions in the aortic wall. She underwent an AVR with a Solo
Smart stentless bioprosthesis (LivaNova PLC, London, UK) (Fig. 2B). She
recovered from the operation well; however, the intermittent
claudication in her calves gradually worsened. Three-dimensional
computed tomography revealed occluded aneurysmal femoropopliteal
arteries with severe calcification (Fig. 2C), which are typical of CALJA
and were similar to the calcifications that had been found in her
sister's lower extremities. She underwent bilateral distal bypass
surgeries for claudication at the age of 73 years.

Currently, both patients are doing well after several cardiac and
vascular operations.

\textbf{Gene analysis}

After filtering the variants between the patients and a non-patient
control (the 76-year-old asymptomatic brother of the patients), we
identified a homozygous variant of the NT5E (5'-ectonucleotidase) gene,
NM\_002526: c.3G\textgreater{}C, (p.Met1?), in the patients (Table 1).
The homozygous variant was not found in the Japanese genome variant
database (HGVD; http://www.hgvd.genome.med.kyoto-u.ac.jp) or the
in-house whole exome sequencing data. The variant was confirmed by
Sanger sequencing. Both patients were genetically diagnosed with CALJA
based on the NT5E mutation.

\textbf{Conclusions}

CALJA is an extremely rare genetic disorder of isolated calcification
that is characterized by late-onset calcification of the arteries and
joints of the extremities. Mutation of the NT5E gene that encodes CD73
have been implicated in CALJA.\textsuperscript{1} CD73 is a
membrane-bound ecto-5'-nucleotidase catalyzing the conversion of AMP to
adenosine.\textsuperscript{1} The enzymic activity of CD73 plays an
important role in the inhibition of inflammation, atherosclerosis, and
ectopic tissue calcification. In patients with this genetic disorder,
ischemic symptoms (including intermittent claudication and chronic
limb-threatening ischemia) are common due to heavily calcified stenotic
or occluded arteries.

Inflammation plays a key role in the pathogenesis of AS regardless of
the presence of anatomical abnormalities. The deletion of the CD73 gene
in mice resulted in almost complete loss of the pathway that degrades
AMP to adenosine at a vascular surface, leading to the development of
aortic valve dysfunction.\textsuperscript{7} The NT5E gene mutation may
also contribute to the pathogenesis of calcific aortic valve disease and
is associated with an increased risk of AS.

We described the cases of two Japanese sisters aged 81 and 74 years,
both of whom underwent AVR for AS and were diagnosed with CALJA and NT5E
gene mutation. These are the first two reported cases of patients with
the NT5E mutation who underwent surgical AVR for AS. The severity of AS
in these patients was similar to that of genetically normal patients
with AS despite the high-risk nature of aortic valve calcification.

Because the symptoms of the NT5E mutation have a late onset, other
factors may also contribute to the severity and timing of CALJA. There
may be undiagnosed patients with the NT5E mutation and CALJA, and the
incidence of homozygous variants of NT5E may be underestimated. Genetic
experts, vascular specialists, and cardiovascular surgeons should
consider the possibility of NT5E mutations in patients who require AVR
for AS and distal arterial bypass surgery for limb ischemia.



\textbf{Acknowledgments and Disclosures:}

We thank Editage (\emph{www.editage.jp}) for English language editing.

\textbf{Disclosures:} The authors have no conflicts of interest to
declare.

\textbf{Author contributions:}

Tetsuro Uchida: Study design (SD), data collection (DC), manuscript
writing (MW), critical revision (CR), and approval of the manuscript for
submission (AS)

Atsushi Yamashita: AS, DC

Ai Ishizawa: AS, DC

Mitsuaki Sadahiro: SD, CR, AS

Nobuyoshi Azuma: SD, CR, AS

Tadashi Kaname: SD, CR, AS, genetic analysis

\textbf{References}

1. St Hilarie C, Ziegler SG, Markello TC, Brusco A, Groden C, Gill F, et
al. NT5E mutations and arterial calcifications. N Engl J Med
2011;364:432-42.

2. Ichikawa N, Taniguchi A, Kaneko H, Kawamoto M, Sekita C, Nakajima A,
et al. Arterial calcification due to deficiency of CD73 (ACDC) as one of
Rheumatic disease associated with periarticular calcification. J Clin
Rheumatol 2015;21:216-20.

3. Zhang Z, He JW, Fu WZ, Zhang CQ, Zhang ZL. Calcification of joints
and arteries: second report with novel NT5E mutations and expansion of
the phenotype. J Hum Genet 2015;60:561-4.

4. de Nijs T, Albuisson J, Ockeloen CW, Legrand A, Jeunemailtre X,
Schultze Kool LJ,et al. Isolated arterial calcifications of the lower
extremities: A clue for NT5E mutation. Int J Cardiol 2016;212:248-50.

5. Yoshioka K, Kuroda S, Takahashi K, Sasano T, Furukawa T, Matsumura A.
Calcification of joints and arteries with novel NT5E mutations with
involvement of upper extremity arteries. Vasc Med 2017;22:541-43.

6. Kochan Z, Karbowska J, Gogga P, Kutryb-Zajac B, Slominska EM,
Smolenski RT. Polymorphism in exon 6 of the human NT5E gene is
associated with aortic valve calcification. Nucleosides Nucleotides
Nucleic Acids 2016;35:726-731.

7. Zukowska P, Kutryb-Zajac B, Jasztal A, Toczek M, Zabielska M,
Borkowski T, et al. Deletion of CD73 in mice leads to aortic valve
dysfunction. Biochim Biophys Acta Mol Basis Dis 2017;1863:1464-72.

8. Sasaki H, Yanagi K, Ugi S, Kobayashi K, Ohkubo K, Tajiri Y, et al.
Definitive diagnosis of mandibular hypoplasia, deafness, progeroid
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mutation in the POLD1 gene. Endocr J 2018;65:227-38.



\textbf{Figure legends}

\textbf{Figure 1: CALJA characteristics in Patient 1} \textbf{a} A
three-dimensional computed tomographic image shows occlusion of the
bilateral femoropopliteal arteries with aneurysmal changes and severe
calcification. The bilateral saphenous vein grafts are patent.\textbf{b}
The patient's aortic valve has severe calcification of the leaflets and
the annulus. \textbf{c} The patient underwent AVR with a bioprosthesis.

AVR: aortic valve replacement; CALJA: calcification of joints and
arteries.

\textbf{Figure 2: CALJA characteristics in Patient 2} \textbf{a} The
patient's aortic valve has severe calcification. \textbf{b} The patient
underwent AVR with a stentless bioprosthesis. \textbf{c} A
three-dimensional computed tomographic image shows occlusion of the
aneurysmal femoropopliteal arteries with dense calcifications.

AVR: aortic valve replacement; CALJA: calcification of joints and
arteries.

\textbf{Table 1: Clinical characteristics and results of the genetic
analyses of both patients}\selectlanguage{english}
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