Interactive CardioVascular and Thoracic Surgery (2021) 1–4 BRIEF COMMUNICATIONS doi:10.1093/icvts/ivab229

Cite this article as: Uchida T, Yamashita A, Ishizawa A, Sadahiro M, Azuma N, Kaname T. NT5E mutation in sisters who underwent aortic valve replacements for aortic stenosis. Interact CardioVasc Thorac Surg 2021; doi:10.1093/icvts/ivab229.

NT5E mutation in sisters who underwent aortic valve replacements

for aortic stenosis ADULT CARDIAC

Tetsuro Uchida a,*, Atsushi Yamashitaa,AiIshizawaa, Mitsuaki Sadahiro a, Nobuyoshi Azumab and Downloaded from https://academic.oup.com/icvts/advance-article/doi/10.1093/icvts/ivab229/6357319 by guest on 28 September 2021 Tadashi Kaname c a Department of Cardiovascular, Thoracic and Paediatric Surgery, Faculty of Medicine, Yamagata University, Yamagata, Japan b Department of Vascular Surgery, Asahikawa Medical University, Asahikawa, Japan c Department of Genome Medicine, National Centre for Child Health and Development, Tokyo, Japan

* Corresponding author. Department of Cardiovascular, Thoracic and Paediatric Surgery, Faculty of Medicine, Yamagata University, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan. Tel: +81-23-6285342; fax: +81-23-6285345; e-mail: [email protected] (T. Uchida).

Received 28 May 2020; received in revised form 12 July 2021; accepted 25 July 2021

Abstract 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; therefore, its deletion results in aortic valve calcification. However, to date, there are no reports of a patient with CALJA with aortic stenosis. In this study, we describe 2 extremely rare cases of sisters with identical NT5E gene mutation patterns, both of whom developed late-onset severe aortic stenosis and limb ischaemia. Both patients underwent aortic valve replacement and bilateral distal arterial bypass surgeries successfully. They were genetically diagnosed with CALJA based on the NT5E mutation. Our report suggests that NT5E mutations should be considered in patients requiring aortic valve replacement for a calcified aortic valve and bypass surgery for specific calcified and occluded arteries. Keywords: NT5E gene mutation • Aortic stenosis • Aortic valve replacement

INTRODUCTION PATIENTS AND METHODS

In 2011, mutation of the NT5E gene that encodes a cluster of dif- Both sisters underwent AVR for AS and distal bypass surgeries for ferentiation 73 (CD73) was found to result in specific calcifica- chronic limb-threatening ischaemia. As they presented with typi- tions in patients with aneurysmal dilatation of arteries as well as cal features of CALJA, both patients underwent a genetic exami- joint calcifications. These symptoms were termed calcification of nation for a definitive diagnosis of CALJA. joints and arteries (CALJA) [1]. Since then, only 14 patients from 7 Genomic DNA was extracted from whole blood cells using a families have been reported as having CALJA [2–5]. Patients with QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). We per- CALJA often experience ischaemic symptoms in the lower ex- formed whole-exome sequencing analysis as previously described [8]. tremities secondary to severely calcified and occluded arteries. In brief, we used the SureSelect Human All Exon V6 kit (Agilent CD73 is also known to play a protective role against aortic valve Technology, Santa Clara, CA, USA) for capture and a HiSeq2500 calcification by hydrolyzation of adenosine monophosphate (Illumina, San Diego, CA, USA) for sequencing. Reads were aligned to (AMP) to adenosine [6]. Although the deletion of the CD73 gene GRC37 using Burrows-Wheeler Aligner (http://bio-bwa.sourceforge. has been reported to cause aortic valve calcification leading to net/). Variants were identified using the GATK Unified Genotyper and valvular stenosis in animal models [7], there are no reports of ANNOVA (http://annover.openbioinfomatics.org/en/latest/). patients with CALJA requiring aortic valve replacement (AVR) for Genetic analysis of the patients was approved by the ethics aortic stenosis (AS). We present an extremely rare case of 2 sisters committee of Yamagata University. Both patients and their family with identical NT5E gene mutation patterns, both of whom members provided informed consent for clinical and genetic underwent AVR for AS. examinations and the publication of this report.

Parts of these observations have been published before and can be found under https://www.authorea.com/ with the doi: 10.22541/au.159007760.01587373.

VC The Author(s) 2021. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved. 2 T. Uchida et al. / Interactive CardioVascular and Thoracic Surgery

RESULTS aortic valve stenosis with a peak pressure gradient of 90 mmHg. Echocardiographic findings and the timing of AVR and distal by- Patient 1 pass surgeries are shown in the timeline (Fig. 2B). She also had a history of bilateral intermittent claudication of the calves and A 77-year-old woman presented with exertional fatigue. She had a chronic coldness of her lower extremities. Surgical treatment of 50-year history of joint pain in her legs and underwent bilateral AS was planned. Her native aortic valve was tricuspid, with severe distal bypass surgeries at 72 years of age for intermittent claudica- calcification of the cusps and annulus (Fig. 1D). There were no tion. Plain radiography of hands showed metacarpal calcification calcified lesions in the aortic wall. She underwent an AVR with a and enlargement of bone ends (Fig. 1A). Plain radiography of the Solo Smart stentless bioprosthesis (LivaNova PLC, London, UK). lower extremities revealed significant calcification with areas of She recovered from the operation well; however, the intermittent Downloaded from https://academic.oup.com/icvts/advance-article/doi/10.1093/icvts/ivab229/6357319 by guest on 28 September 2021 arteriomegaly. Three-dimensional computed tomography revealed claudication in her calves gradually worsened. Three-dimensional extensive occlusion of bilateral femoropopliteal arteries with aneu- computed tomography revealed occluded aneurysmal femoro- rysmal changes and severe calcifications, which are typical findings popliteal arteries with severe calcification (Fig. 1E), which are typ- of CALJA (Fig. 1B). The patient’s saphenous vein grafts were patent ical of CALJA, and was similar to the calcifications that had been when she presented to us. During her recovery from the vascular found in her sister’s lower extremities. She underwent bilateral reconstructive surgeries, the patient gradually developed exertional distal bypass surgeries for claudication at the age of 73 years. She fatigue. Echocardiography revealed severe aortic valve stenosis is in good condition 38 months after AVR. with a peak pressure gradient of 50 mmHg and AVR was planned. Currently, both patients are doing well after several cardiac Echocardiographic findings and the timing of AVR and distal by- and vascular operations. pass surgeries are shown in the timeline (Fig. 2A). The native aortic valve was tricuspid, and the valvular leaflets and annulus showed GENE ANALYSIS significant calcification (Fig. 1C). However, no calcification was ob- served in the aortic wall. She underwent AVR with a Carpentier- After filtering the variants between the patients and a non- Edwards PERIMOUNT bioprosthetic valve (Edwards Lifesciences, patient control (the 76-year-old asymptomatic brother of the Irvine, CA, USA). Her postoperative course was uneventful and she patients), we identified a homozygous variant of the NT5E (50- is doing well 50 months after AVR. ectonucleotidase) gene, NM_002526: c.3G>C (p.Met1?). The ho- mozygous variant was not found in the Japanese genome variant Patient 2 database (HGVD; http://www.hgvd.genome.med.kyoto-u.ac.jp)or the in-house whole-exome sequencing data. The variant was A 71-year-old woman, who is the younger sister of patient 1, had confirmed by Sanger sequencing. Both patients were genetically developed dyspnoea on exertion and was diagnosed with severe diagnosed with CALJA based on the NT5E mutation.

Figure 1: Calcification of joints and arteries characteristics in patients. (A) Plain radiography of left hands shows metacarpal calcification (arrowhead) and enlargement of bone ends (dotted circle). (B) A 3-dimensional computed tomographic image shows occlusion of the bilateral femoropopliteal arteries with aneurysmal changes and severe calcification. The bilateral saphenous vein grafts are patent. (C) The patient’s aortic valve has severe calcification of the leaflets and the annulus. (D) The patient’s aortic valve has severe calcification. (E) A 3-dimensional computed tomographic image shows occlusion of the aneurysmal femoropopliteal arteries with dense calcification. T. Uchida et al. / Interactive CardioVascular and Thoracic Surgery 3 ADULT CARDIAC Downloaded from https://academic.oup.com/icvts/advance-article/doi/10.1093/icvts/ivab229/6357319 by guest on 28 September 2021

Figure 2: Timeline of surgical treatment in both patients and echocardiographic findings. (A) Patient 1. (B) Patient 2. AVA: aortic valve area; AVR: aortic valve replace- ment; PG: pressure gradient.

DISCUSSION factors might contribute to the timing of disease onset and its se- verity. The exact relationship between the NT5E mutation and CALJA is an extremely rare genetic disorder of isolated calcifica- the late-onset of both the aortic valve and peripheral arterial dis- tion that is characterized by late-onset calcification of the arteries ease is to be elucidated in the future. and joints of the extremities. Mutation of the NT5E gene that We described the cases of 2 Japanese sisters aged 77 and encodes CD73 has been implicated in CALJA [1]. CD73 is a 71 years, both of whom underwent AVR for AS and were diag- membrane-bound ecto-50-nucleotidase catalysing the conversion nosed with CALJA and NT5E gene mutation. These are the first 2 of AMP to adenosine [1]. The enzymic activity of CD73 plays an reported cases of patients with the NT5E mutation who under- important role in the inhibition of inflammation, atherosclerosis, went surgical AVR for AS. The severity of AS in these patients was and ectopic tissue calcification. In patients with this genetic disor- similar to that of genetically normal patients with AS despite the der, ischaemic symptoms (including intermittent claudication high-risk nature of aortic valve calcification. and chronic limb-threatening ischaemia) are common due to Currently, the NT5E mutation and CALJA are not widely known heavily calcified stenotic or occluded arteries. in the surgical field. Therefore, there may be undiagnosed Inflammation plays a key role in the pathogenesis of AS re- patients with this rare genetic disorder, and the incidence of ho- gardless of the presence of anatomical abnormalities. The dele- mozygous variants of NT5E may be underestimated. Genetic tion of the CD73 gene in mice resulted in almost complete loss experts, vascular specialists, and cardiovascular surgeons should of the pathway that degrades AMP to adenosine at a vascular consider the possibility of NT5E mutations in patients who re- surface, leading to the development of aortic valve dysfunction quire AVR for AS and distal arterial bypass surgery for limb [7]. The NT5E gene mutation may also contribute to the patho- ischaemia. genesis of calcific aortic valve disease and is associated with an increased risk of AS. The CD73 activity is essential for the normal aortic valve function and the preservation of its structure. CONCLUSION In animal experiments, the stenotic aortic valve dysfunction of CD73 knockout mice fed a normal-fat diet are reported to be In the study, we presented and identified 2 extremely rare cases more progressive and severe than wild-type mice fed a high-fat of sisters with identical NT5E gene mutation patterns, both of diet, which is one of the established models of aortic valve dis- whom underwent AVR for AS. ease [7]. Furthermore, it should be noted that CD73 knockout mice fed in a high-fat showed the highest peak aortic valve flow velocity and smallest aortic valve area in the experimental groups [7]. These results may suggest that CD73 deletion will lead to ex- ACKNOWLEDGEMENTS acerbation of a pre-existed age-related aortic valve disease due to hyperlipidaemia and other cardiovascular risk factors. The authors thank Editage (www.editage.jp) for English language The reason why both aortic valvular and peripheral arterial dis- editing. eases are late-onset and progression and severity of the periph- eral disease is worse than aortic valve disease are still unknown since only a few cases have been reported. Various acquired Conflict of interest: none declared. 4 T. Uchida et al. / Interactive CardioVascular and Thoracic Surgery

Author contributions [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 Tetsuro Uchida: Conceptualization; Data curation; Investigation; Rheumatol 2015;21:216–20. Writing—original draft. Atsushi Yamashita: Data curation. Ai [3] Zhang Z, He JW, Fu WZ, Zhang CQ, Zhang ZL. Calcification of joints and Ishizawa: Data curation. Mitsuaki Sadahiro: Supervision. arteries: second report with novel NT5E mutations and expansion of the Nobuyoshi Azuma: Conceptualization; Supervision. Tadashi phenotype. J Hum Genet 2015;60:561–4. Kaname: Conceptualization; Investigation; Supervision. [4] de Nijs T, Albuisson J, Ockeloen CW, Legrand A, Jeunemaitre 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. Reviewer information Calcification of joints and arteries with novel NT5E mutations with in- Downloaded from https://academic.oup.com/icvts/advance-article/doi/10.1093/icvts/ivab229/6357319 by guest on 28 September 2021 volvement of upper extremity arteries. Vasc Med 2017;22:541–3. Interactive CardioVascular and Thoracic Surgery thanks Lucio Cagini [6] Kochan Z, Karbowska J, Gogga P, Kutryb-Zajac B, Slominska EM, and the other, anonymous reviewer(s) for their contribution to the Smolenski RT. Polymorphism in exon 6 of the human NT5E gene is asso- peer review process of this article. ciated with aortic valve calcification. Nucleosides Nucleotides Nucleic Acids 2016;35:726–31. [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. REFERENCES 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. [1] St Hilaire C, Ziegler SG, Markello TC, Brusco A, Groden C, Gill F et al. Definitive diagnosis of mandibular hypoplasia, deafness, progeroid fea- NT5E mutations and arterial calcifications. N Engl J Med 2011;364: tures and lipodystrophy (MDPL) syndrome caused by a recurrent de 432–42. novo mutation in the POLD1 gene. Endocr J 2018;65:227–38.