Association between Severity of Clinical Impairment and Intensity of Dermal Histological Lesions in Pseudoxanthoma Elasticum

(1)

Membres du jury

Pr ROUSSELET Marie-Christine | Présidente Pr MARTIN Ludovic | Directeur Pr BONNEAU Dominique | Membre

Dr CROUE Anne | Membre Dr ZIEGLER Alban | Membre

Soutenue publiquement le :

06 juillet 2020

2019-2020

THÈSE

pour le

DIPLÔME D’ÉTAT DE DOCTEUR EN MÉDECINE Qualification en DERMATOLOGIE

ASSOCIATION BETWEEN SEVERITY OF CLINICAL IMPAIRMENT AND

INTENSITY OF DERMAL HISTOLOGICAL LESIONS IN PSEUDOXANTHOMA ELASTICUM

Association entre la sévérité de l’atteinte clinique et l’intensité des lésions histologiques dermiques dans le

pseudoxanthome élastique

DELAITRE Léa

Né le 27 décembre 1992 à Paris XIX (75)

Sous la direction du Professeur MARTIN Ludovic

(2)

(3)

ENGAGEMENT DE NON PLAGIAT

Je, soussignée Léa DELAITRE déclare être pleinement consciente que le plagiat de documents ou d’une partie d’un document publiée sur toutes formes de support, y compris l’internet, constitue une violation des droits d’auteur ainsi qu’une fraude caractérisée.

En conséquence, je m’engage à citer toutes les sources que j’ai utilisées pour écrire ce rapport ou mémoire.

signée par l'étudiante le 23/01/2020

(4)

LISTE DES ENSEIGNANTS DE LA FACULTÉ DE SANTÉ D’ANGERS

Doyen de la Faculté : Pr Nicolas Lerolle

Vice-Doyen de la Faculté et directeur du département de pharmacie : Pr Frédéric Lagarce

Directeur du département de médecine : Pr Cédric Annweiler PROFESSEURS DES UNIVERSITÉS

ABRAHAM Pierre Physiologie Médecine

ANNWEILER Cédric Gériatrie et biologie du vieillissement Médecine

ASFAR Pierre Réanimation Médecine

AUBE Christophe Radiologie et imagerie médicale Médecine

AUGUSTO Jean-François Néphrologie Médecine

AZZOUZI Abdel Rahmène Urologie Médecine

BAUFRETON Christophe Chirurgie thoracique et

cardiovasculaire Médecine

BENOIT Jean-Pierre Pharmacotechnie Pharmacie

BEYDON Laurent Anesthésiologie-réanimation Médecine

BIGOT Pierre Urologie Médecine

BONNEAU Dominique Génétique Médecine

BOUCHARA Jean-Philippe Parasitologie et mycologie Médecine

BOUVARD Béatrice Rhumatologie Médecine

BOURSIER Jérôme Gastroentérologie ; hépatologie Médecine

BRIET Marie Pharmacologie Médecine

CAILLIEZ Eric Médecine générale Médecine

CALES Paul Gastroentérologe ; hépatologie Médecine CAMPONE Mario Cancérologie ; radiothérapie Médecine CAROLI-BOSC François-xavier Gastroentérologie ; hépatologie Médecine CHAPPARD Daniel Cytologie, embryologie et

cytogénétique Médecine

CONNAN Laurent Médecine générale Médecine

COUTANT Régis Pédiatrie Médecine

CUSTAUD Marc-Antoine Physiologie Médecine

DE CASABIANCA Catherine Médecine Générale Médecine DESCAMPS Philippe Gynécologie-obstétrique Médecine D’ESCATHA Alexis Médecine et santé au Travail Médecine DINOMAIS Mickaël Médecine physique et de

réadaptation Médecine

DIQUET Bertrand Pharmacologie Médecine

DUBEE Vincent Maladies Infectieuses et Tropicales Médecine DUCANCELLE Alexandra Bactériologie-virologie ; hygiène

hospitalière Médecine

DUVAL Olivier Chimie thérapeutique Pharmacie

DUVERGER Philippe Pédopsychiatrie Médecine

(5)

EVEILLARD Mathieu Bactériologie-virologie Pharmacie FAURE Sébastien Pharmacologie physiologie Pharmacie

FOURNIER Henri-Dominique Anatomie Médecine

FURBER Alain Cardiologie Médecine

GAGNADOUX Frédéric Pneumologie Médecine

GARNIER François Médecine générale Médecine

GASCOIN Géraldine Pédiatrie Médecine

GOHIER Bénédicte Psychiatrie d'adultes Médecine

GUARDIOLA Philippe Hématologie ; transfusion Médecine

GUILET David Chimie analytique Pharmacie

HAMY Antoine Chirurgie générale Médecine

HENNI Samir Chirurgie Vasculaire, médecine

vasculaire Médecine

HUNAULT-BERGER Mathilde Hématologie ; transfusion Médecine

IFRAH Norbert Hématologie ; transfusion Médecine

JEANNIN Pascale Immunologie Médecine

KEMPF Marie Bactériologie-virologie ; hygiène

LACCOURREYE Laurent Oto-rhino-laryngologie Médecine

LAGARCE Frédéric Biopharmacie Pharmacie

LARCHER Gérald Biochimie et biologie moléculaires Pharmacie LASOCKI Sigismond

LEGENDRE Guillaume Anesthésiologie-réanimation

Gynécologie-obstétrique Médecine Médecine

LEGRAND Erick Rhumatologie Médecine

LERMITE Emilie Chirurgie générale Médecine

LEROLLE Nicolas Médecine Intensive-Réanimation Médecine LUNEL-FABIANI Françoise Bactériologie-virologie ; hygiène

MARCHAIS Véronique Bactériologie-virologie Pharmacie

MARTIN Ludovic Dermato-vénéréologie Médecine

MAY-PANLOUP Pascale Biologie et médecine du développement et De la reproduction

Médecine

MENEI Philippe Neurochirurgie Médecine

MERCAT Alain Réanimation Médecine

MERCIER Philippe Anatomie Médecine

PAPON Nicolas Parasitologie et mycologie médicale Pharmacie

PASSIRANI Catherine Chimie générale Pharmacie

PELLIER Isabelle Pédiatrie Médecine

PETIT Audrey Médecine et Santé au Travail Médecine PICQUET Jean Chirurgie vasculaire ; médecine

vasculaire Médecine

PODEVIN Guillaume Chirurgie infantile Médecine

PROCACCIO Vincent Génétique Médecine

PRUNIER Delphine Biochimie et Biologie Moléculaire Médecine

PRUNIER Fabrice Cardiologie Médecine

REYNIER Pascal Biochimie et biologie moléculaire Médecine

(6)

RICHARD Isabelle Médecine physique et de

réadaptation Médecine

RICHOMME Pascal Pharmacognosie Pharmacie

RODIEN Patrice Endocrinologie, diabète et maladies

métaboliques Médecine

ROQUELAURE Yves Médecine et santé au travail Médecine ROUGE-MAILLART Clotilde Médecine légale et droit de la santé Médecine ROUSSEAU Audrey Anatomie et cytologie pathologiques Médecine ROUSSEAU Pascal Chirurgie plastique, reconstructrice

et esthétique Médecine

ROUSSELET Marie-Christine Anatomie et cytologie pathologiques Médecine

ROY Pierre-Marie Thérapeutique Médecine

SAULNIER Patrick Biophysique et biostatistique Pharmacie

SERAPHIN Denis Chimie organique Pharmacie

TRZEPIZUR Wojciech Pneumologie Médecine

UGO Valérie Hématologie ; transfusion Médecine

URBAN Thierry Pneumologie Médecine

VAN BOGAERT Patrick Pédiatrie Médecine

VENIER-JULIENNE Marie-Claire Pharmacotechnie Pharmacie

VERNY Christophe Neurologie Médecine

WILLOTEAUX Serge Radiologie et imagerie médicale Médecine

MAÎTRES DE CONFÉRENCES

ANGOULVANT Cécile Médecine Générale Médecine

BAGLIN Isabelle Chimie thérapeutique Pharmacie

BASTIAT Guillaume Biophysique et biostatistique Pharmacie

BEAUVILLAIN Céline Immunologie Médecine

BELIZNA Cristina Médecine interne Médecine

BELLANGER William Médecine générale Médecine

BELONCLE François Réanimation Médecine

BENOIT Jacqueline Pharmacologie Pharmacie

BIERE Loïc Cardiologie Médecine

BLANCHET Odile Hématologie ; transfusion Médecine

BOISARD Séverine Chimie analytique Pharmacie

CAPITAIN Olivier Cancérologie ; radiothérapie Médecine

CASSEREAU Julien Neurologie Médecine

CHAO DE LA BARCA Juan-Manuel Médecine

CHEVALIER Sylvie Biologie cellulaire Médecine

CLERE Nicolas Pharmacologie / physiologie Pharmacie

COLIN Estelle Génétique Médecine

DERBRE Séverine Pharmacognosie Pharmacie

DESHAYES Caroline Bactériologie virologie Pharmacie

FERRE Marc Biologie moléculaire Médecine

FORTRAT Jacques-Olivier Physiologie Médecine

HAMEL Jean-François Biostatistiques, informatique médicale Médicale

(7)

HINDRE François Biophysique Médecine KHIATI Salim Biochimie et biologie moléculaire Médecine JOUSSET-THULLIER Nathalie Médecine légale et droit de la santé Médecine JUDALET-ILLAND Ghislaine Médecine Générale Médecine KUN-DARBOIS Daniel Chirurgie Maxillo-Faciale et

Stomatologie Médecine

LACOEUILLE Franck Biophysique et médecine nucléaire Médecine

LEBDAI Souhil Urologie Médecine

LANDREAU Anne Botanique/ Mycologie Pharmacie

LEBDAI Souhil Urologie Médecine

LEGEAY Samuel Pharmacocinétique Pharmacie

LE RAY-RICHOMME Anne-

Marie Pharmacognosie Pharmacie

LEPELTIER Elise Chimie générale Pharmacie

LETOURNEL Franck Biologie cellulaire Médecine

LIBOUBAN Hélène Histologie Médecine

LUQUE PAZ Damien Hématologie; Transfusion Médecine

MABILLEAU Guillaume Histologie, embryologie et

cytogénétique Médecine

MALLET Sabine Chimie Analytique Pharmacie

MAROT Agnès Parasitologie et mycologie médicale Pharmacie

MESLIER Nicole Physiologie Médecine

MOUILLIE Jean-Marc Philosophie Médecine

NAIL BILLAUD Sandrine Immunologie Pharmacie

PAILHORIES Hélène Bactériologie-virologie Médecine

PAPON Xavier Anatomie Médecine

PASCO-PAPON Anne Radiologie et imagerie médicale Médecine

PECH Brigitte Pharmacotechnie Pharmacie

PENCHAUD Anne-Laurence Sociologie Médecine

PIHET Marc Parasitologie et mycologie Médecine

PY Thibaut Médecine Générale Médecine

RAMOND-ROQUIN Aline Médecine Générale Médecine

RINEAU Emmanuel Anesthésiologie réanimation Médecine

RIOU Jérémie Biostatistiques Pharmacie

ROGER Emilie Pharmacotechnie Pharmacie

SAVARY Camille Pharmacologie-Toxicologie Pharmacie

SCHMITT Françoise Chirurgie infantile Médecine

SCHINKOWITZ Andréas Pharmacognosie Pharmacie

SPIESSER-ROBELET

Laurence Pharmacie Clinique et Education

Thérapeutique Pharmacie

TANGUY-SCHMIDT Aline TESSIER-CAZENEUVE Christine

Hématologie ; transfusion

Médecine Générale Médecine

Médecine

VENARA Aurélien Chirurgie générale Médecine

VIAULT Guillaume Chimie organique Pharmacie

(8)

PROFESSEURS EMERITES

Philippe MERCIER Neurochirurgie Médecine

Dominique CHABASSE Parasitologie et Médecine Tropicale Médecine

Jean-François SUBRA Néphrologie Médecine

AUTRES ENSEIGNANTS

AUTRET Erwan Anglais Médecine

BARBEROUSSE Michel Informatique Médecine

BRUNOIS-DEBU Isabelle Anglais Pharmacie

CHIKH Yamina Économie-Gestion Médecine

FISBACH Martine Anglais Médecine

O’SULLIVAN Kayleigh Anglais Médecine

PAST

CAVAILLON Pascal Pharmacie Industrielle Pharmacie

LAFFILHE Jean-Louis Officine Pharmacie

MOAL Frédéric Pharmacie clinique Pharmacie

ATER

KILANI Jaafar Biotechnologie Pharmacie

WAKIM Jamal Biochimie et chimie biomoléculaire Médecine

(9)

REME RC IEM ENTS

Aux membres du jury de thèse,

 Au Professeur Marie-Christine ROUSSELET,

Vous me faites l’honneur de présider mon jury de thèse. Merci pour votre accueil dans le service d’anatomo-pathologie. Soyez assurée de mon profond respect.

 Au Professeur Ludovic MARTIN,

Merci pour votre implication dans notre enseignement durant tout l’internat et pour l’encadrement de cette thèse. Soyez assuré de ma profonde reconnaissance.

Au Docteur Anne CROUE,

Merci de me faire l’honneur de participer à l’évaluation de mon travail. J’ai beaucoup apprécié mon semestre dans le service d’anatomo-pathologie où j’ai pu découvrir cette spécialité passionnante et complexe.

 Au Docteur Alban ZIEGLER,

Merci d’avoir accepté de faire partie de ce jury de thèse. Veuillez trouver ici mes sincères remerciements.

 Au Professeur Dominique BONNEAU,

Merci de me faire l’honneur de participer à mon jury.

Je remercie l’ensemble des équipes médicales et paramédicales qui m’ont accueillie durant mon internat.

 Aux dermatologues qui m’ont tant apporté durant ma formation,

Aux Docteurs Yannick LE CORRE, Christian LE CLEC’H, Solène RUIZ, Xavier GRIMAUX, Caroline BEAUCHENE, Juliette DELAUNAY, Hervé MAILLARD, Corina BARA, Nathalie BENETON, Blandine VINCENEUX, Joana MARTINS-HERICHER. Je vous remercie sincèrement pour vos enseignements.

(10)

REME RC IEM ENTS

 Aux infectiologues du CH du Mans,

Je vous remercie pour votre accueil, votre confiance, votre gentillesse, et votre encadrement durant mes premiers pas en tant qu’interne.

 Aux médecins et internes du service d’anatomo-pathologie :

Pour votre accueil dans le service, la découverte de cette spécialité très intéressante et les staffs très pédagogiques.

A mes cointernes Baptiste, Théo, Alex et Benjamin, pour ce semestre sympa en votre compagnie.

 Aux chirurgiens plastiques,

Pour la découverte de votre spécialité et la bonne ambiance dans ce service. J’ai beaucoup appris à vos côtés.

 Aux Docteurs VANDAMME, PAJOT, GUERIN-MOREAU et MANCEL, Merci pour votre accueil et votre enseignement durant mon stage en libéral.

 Aux étudiants de Master 1 (Xavier, Hugo, Annaëlle, Marine, Manon, Helena, Emmanuel, Chloé, Adèle), Merci pour votre travail précieux.

 A Nastia NAVASIOLAVA,

Merci pour ta disponibilité, ta gentillesse, ton précieux travail et la tenue rigoureuse de la base de données.

 A Patrick Saulnier, pour votre aide précieuse pour les statistiques.

(11)

REME RC IEM ENTS

 A mes cointernes de dermato,

Notamment Diane avec qui j’étais heureuse de faire tous ces séminaires et 2 semestres; et Mathilde L que j’aurais pu rencontrer bien plus tôt, avec qui j’ai partagé un super semestre et de nombreux covoiturages.

Je suis heureuse de faire partie de la team dermato à vos côtés.

 A mes parents et à mon frère,

Merci pour votre soutien et pour avoir toujours cru en moi.

Merci Maman pour ton amour et ton aide lors de tous les déménagements.

 Aux Cointernes RPZ (Faten, Isabelle, Sabine, Jérémy, Pierre, Julie, Thomas, Marine

A, Polo, Nynou et Nynette, Alisée),

Merci pour tous ces moments passés ensemble depuis le premier semestre. Je suis très heureuse de vous avoir rencontrés. L’internat est passé très vite grâce à vous.

 A mes amies Anna et Céline,

Deux belles rencontres durant l’internat. Anna, j’ai adoré être ta coloc pendant 1 an.

 A Joël,

Pour ton amour et ta présence à mes côtés.

(12)

LIST OF ABBREVIATIONS

PXE Pseudoxanthoma Elasticum PPi Inorganic PyroPhosphate

GACI Generalized Arterial Calcification of Infancy

CT Computer Tomography

LLAC Lower Limb Arterial Calcification SD Standard Deviation

(13)

Plan

LIST OF ABBREVIATIONS ABSTRACT

INTRODUCTION

PATIENTS AND METHODS RESULTS

1. Calcium Scores 1.1. Age

1.2. Sex

1.3. Skin Involvement

1.4. Severe Ophthalmic Impairment 1.5. Cardiovascular Manifestations 1.6. Coronary Calcium Scoring

1.7. Lower Limb Arterial Calcification Scores 1.8. Plasma PPi

2. Area Occupied by Elastic fibers 2.1. Age

2.2. Sex

DISCUSSION CONCLUSION REFERENCES LIST OF FIGURES TABLE OF CONTENTS APPENDICES

(14)

INTRODUCTION

1- Epidemiology

Pseudoxanthoma elasticum (PXE) is a rare systemic metabolic disease with autosomal recessive inheritance (OMIM 264800) caused by mutations in the ABCC6 gene (located on the short arm of chromosome 16) encoding a transmembrane transport protein expressed predominantly in the liver (1). Its estimated prevalence is around 1/40.000, with a female predominance. Over 300 ABCC6 mutations have been identified to date (2). Around 90% of patients with clinical PXE have a mutation in both alleles (3).

2- Clinical Features

PXE is characterized by the calcification and fragmentation of elastic fibers (termed elastorrhexis) mainly affecting the skin, retina and arterial walls (4,5). Diagnosis is based on identification of skin lesions, presence of angioid streaks on funduscopy and fragmented and calcified elastic fibers detected on skin biopsy. This disease displays wide clinical heterogeneity.

Skin Manifestations:

The principal clinical sign of PXE is customarily skin-related. The first manifestations occur in the second decade of life. Small yellowish papules develop initially on lateral aspects of the neck and in flexural areas (axillary, inguinal, antecubital and umbilicus). The papules then slowly coalesce, and the skin becomes loose and inelastic.

Ocular Findings:

Ophthalmic impairment affects the retina, involving mottled pigmentation (peau d’orange), formation of drusen or angioid streaks (tears in the abnormally-calcified Bruch’s membrane of the retina) and neovessels in which bleeding can cause impaired vision, occasionally resulting in bilateral central vision loss. 100% of patients over the age of 20 years present

(15)

2 with angioid streaks (6). Intraocular injections of anti-VEGF play a part in combating choroidal neovascularisation. However, angioid streaks are not necessarily pathognomonic of PXE since they may occur in disorders such as sickle cell disease and thalassemia (7).

Vascular Injury:

The clinical manifestations of vascular injury include lower extremity peripheral artery disease (peripheral pulse loss, intermittent claudication), renovascular hypertension, intestinal ischemia, coronary artery disease resulting in angina pectoris, and more rarely myocardial infarction. Characteristic signs of cerebrovascular disease are transient ischemic attacks, aneurysms, strokes and multi-infarct dementia (5,8–10). Lower extremity peripheral artery disease results from progressive mineralization of the elastic media and intima primarily in the mid-sized arteries. This mineralization is linked to intimal hyperplasia and arterial narrowing. As a general rule, it is the main criterion for disease severity.

Other Clinical Events:

Gastric bleeding complicates 5% of cases, especially during adolescence and early adulthood.

Kidney stones are more common in PXE patients than in the general population. In the Angers PXE cohort, 39.8% of patients declared previous history of kidney stones (11).

3- Histology

Diagnosis of PXE is confirmed by punch skin biopsy. Elastorrhexis and calcification of the elastic fibers in the reticular dermis are visible on histological examination. These features are easily identifiable by von Kossa (calcifications in black) and Verhoeff-van Gieson (elastic fibers in black) staining. Mid-dermal elastic fibers appear short, fragmented, clumped, coiled, thickened and calcified (3,12). Histology can occasionally be used to diagnose patients presenting with angioid streaks but no visible skin lesions (13).

Data on clinicopathological correlation is scarce in the literature. Dermoscopic investigation of PXE conducted by Berthin et al. found histological PXE lesions in an area where skin lesions

(16)

were invisible to the naked eye. Calcium impregnation and elastic fiber fragmentation appeared to be less extensive in these samples than in biopsies of clinically obvious PXE lesions (14). A case study by Persechino of a 17-year-old female patient found a correlation between clinical aspect, dermoscopy, optical coherence tomography and histology (15). Vos et al. (16) investigated a correlation between the histological lesions of two PXE patients and lesions scanned on post-mortem full-body imaging. Many of the subtle PXE-related abnormalities found in other organ systems during autopsy had not been detected on CT scans. CT imaging had found only vascular and skin lesions, whereas post-mortem examination uncovered esophageal, subcutaneous and pleural lesions.

4- Pathophysiology

The pathophysiology of PXE is not yet fully understood. Functional ABCC6 deficiency is associated with a reduction in adenosine triphosphatase secretion in the liver circulation, leading to a 60% decrease in plasma inorganic pyrophosphate (PPi) levels (17–19). PPi is a powerful ectopic mineralization inhibitor (20,21). In PXE, decreased PPi levels can therefore lead to ectopic mineralization (18). It remains to be determined whether, and to what extent, other issues (vitamin K deficiency etc.) also play a part in lowering PPi levels, thereby causing ectopic calcification (22–24).

5- Therapy

No etiological treatment has to date been shown to effectively combat PXE, but research into PXE and related ectopic mineralization disorders is gaining ground (25). Patient management is complex and is essentially based on prevention of and screening for ophthalmic and cardiovascular complications. Patients are advised to abstain from high-risk sports susceptible to cause ocular trauma and are encouraged to make improvements to their diet and lifestyle to delay cardiovascular complications.

(17)

4 Studies are in progress to assess the efficacy of magnesium supplementation or, with greater prospects for success, etidronate therapy in PXE patients. Modulating dietary PPi is a potentially effective approach for preventing or reducing calcification in ABCC6^-/- mice (26,27). Unfortunately, PPi is extremely labile and has a short half-life due to the effect of hydrolysis on this molecule. As a result the possibility of using stable, non-hydrolysable pyrophosphate analogues – bisphosphonates – has been evoked in the treatment of ectopic mineralization disorders. The bisphosphonate etidronate is a molecular substitute for PPi.

Treatment with etidronate significantly reduces arterial calcification and subretinal neovascularization compared to placebo in ABCC6^-/- mice (28). Etidronate has been shown to halt the progression of calcification in all PXE patient vascular beds excepting the coronary arteries (29). Further research is required to assess the long-term safety and efficacy of etidronate with respect to clinical outcomes and generalizability.

6- Study Rationale and Objectives

The severity of skin, cardiovascular, ophthalmic and histological damage is variable from one subject to another. At present there is no basis (in terms of histology for instance) for predicting the clinical course of organ damage in a given patient.

A link was found in two studies between number of affected skin sites and development of cardiovascular or ophthalmic complications (30,31). This begs the question as to whether there is an association between dermal lesions and organ damage.

The aim of the present study was to investigate a correlation between the severity of clinical manifestations in PXE patients and the severity of dermal lesions assessed histologically in the skin biopsies obtained from these patients. To this end, we elaborated assessment tools for measuring calcification and elastic fibers on histology slides.

(18)

METHODS

The present study focused on a cohort of PXE patients from the Angers University Hospital PXE Specialist Center (France). The PXE patient database monitored at Angers Hospital is composed of 183 patients (Figure 1) who received a clear-cut diagnosis between 2008 and 2019. These patients provided informed consent for the collection of clinical and biological data (ClinicalTrials register NCT01446380). They attend appointments every two to three years in the absence of complications to assess their condition via: biological blood and urine tests, cardiac examination, skin biopsy where necessary, arterial Doppler ultrasound imaging of the lower limb arteries, transthoracic echocardiography, and occasionally coronary angiography imaging or ultrasonography of the lower limb arteries. Clinical and biological patient characteristics are recorded in an anonymized database.

Inclusion criteria for this study were:

- Patients with indisputable PXE (clinically and histologically compatible aspects)

- Patients admitted to Angers University Hospital for assessment of PXE status between 2008 and 2019

- Skin biopsies obtained from the antecubital fossa. The antecubital fossa is seldom exposed to the sun and was the preferred site for pathological diagnosis in our patient group. Any over-estimation of PXE-induced elastorrhexis is thus avoided by taking actinic elastosis into account. Use of a single skin area ensures good measurement reproducibility. The percentage area occupied by calcification or fragmented elastic fibers likely varies depending on the dermal thickness of the area under investigation.

- Usable histological sections. Histological sections with artifacts (significant tears or staining artifacts) were excluded.

(19)

6 Histological slides used in von Kossa staining (to visualize calcification) and in Verhoeff staining (to visualize elastic fibers) were photographed using an Olympus BX43 microscope with a DP74 camera. The images obtained were in PNG format.

We have developed a reliable and reproducible technique for measuring the area occupied by calcification on skin biopsy imaging. ImageJ software (https://imagej.nih.gov/ij/) was used for image analysis to determine the area occupied by calcification and by fragmented elastic fibers over an area of 700 x 700µm. This area had been determined beforehand to ensure that the majority of the dermis and not the epidermis would be taken into account for measuring. The results were expressed as percentages per area analyzed. To ensure reproducibility, the areas were measured in triplicate. The mean of the three measurements was used.

The procedure followed to calculate the area occupied by calcification using ImageJ software is detailed in Appendix 1.

The procedure followed to calculate the area occupied by calcified elastic fibers is detailed in Appendix 2.

The extent of skin involvement was estimated by enumerating the affected skin sites, and taking into consideration the ten areas most typically affected in the majority of PXE patients (lateral neck, anterior neck, nape, axillary fossae, shoulder joint region, antecubital fossae, groin, popliteal fossae, periumbilical region, lower lip mucosa), and the nine non-typical areas (oblique mental creases, face, upper limbs excluding folds, lower limbs excluding folds, lumbar, genital, anal, oral mucosa excluding lower lip, other areas).

The number of affected skin areas was coded from 0 to 19 (an area was regarded as affected even in the event of unilateral lesions). In our study, we coded presence (1) or absence (0) of skin lesions, regardless of their severity. Severity of skin involvement at the biopsy site

(20)

was coded as follows: 0, no visible lesions; 1, yellowish network; 2, yellowish papules; 3, confluent papules in plaques; 4, loss of skin elasticity.

Severe ophthalmic impairment was defined in our study as unilateral or bilateral vision loss linked to a specific complication of PXE.

Severe cardiovascular disease was defined in our study as coronary artery disease with a history of myocardial infarction or angioplasty and/or a stroke (or transient ischemic attack) and/or revascularized disease of the lower limb arteries.

Calcium scores, adapted from the Agatston scoring system (Berman et al., 2016), generated figures that reflected the extent of calcified atheromatous deposits present within the coronary artery walls. A zero score corresponded to absence of calcified deposits within the entire coronary artery network. This score was obtained from computed tomography (CT) imaging without injection. On the same principle, lower limb arterial calcification scores were calculated using CT imaging of the lower limbs.

Statistical data analysis was conducted on the http://biostatgv.sentiweb.fr website. Non- parametric tests were applied in addition to the Pearson test. The Kruskal-Wallis test was used to estimate the number of affected skin sites. The Spearman test was used for other variables. Trend lines were drawn to illustrate these results.

(21)

8

RESULTS

This retrospective study involved 59 patients treated at the Angers University Hospital Specialist Center for Rare Diseases between 2008 and 2019 for whom clinical data and histological slides were accessible. 59 usable slides were obtained from PXE patient antecubital biopsies using von Kossa and Verhoeff-van Gieson staining.

Figure 1. Flow chart

(22)

Mean patient age was 47.9 years at time of skin biopsy (range: 20-79 years). The cohort comprised 17 men (29%) and 42 women (71%).

1. Calcium Scores

Mean percentage area occupied by calcification was 16.43% (SD=11.8).

The distribution of the areas occupied by calcification did not follow a normal distribution pattern.

1.1 Age

There was no correlation between age and area occupied by calcification (Spearman ρ = 0.04, p=0.76)(Figure 2).

Figure 2. Area occupied by calcification in terms of age.

1.2 Sex

There was no statistically significant difference in area occupied by calcification between men and women (p=0.38).

(23)

10 1.3 Skin Involvement

A mean of 8 affected skin sites was identified in the present study. Correlation between number of affected cutaneous sites and area occupied by calcification was moderate (Spearman ρ=0.35, p<0.01)(Figure 3).

Figure 3: Positive correlation between area occupied by calcification and number of affected skin sites.

Calcification rates are potentially predictive of total affected skin sites greater than or equal to 9 (Figure 4).

(24)

Figure 4: ROC curve for number of affected skin sites ≥ 9. Area under curve for calcification:

0.72 (CI95=0.59-0.85). Area under curve for elastic fibers: 0.63 (CI95=0.48-0.77).

Mean severity of skin involvement at the biopsy site was 1.99. There was a statistically significant difference between the medians (p<0.01) (Figure 5). Moderate correlation was found between severity of skin involvement and area occupied by calcification (Spearman ρ=0.47, p<0.01).

Figure 5: Box plots. Percentage area occupied by calcification in terms of severity of skin involvement (0: no visible lesion; 1: yellowish network; 2: yellowish papules; 3: confluent papules in plaques; 4: loss of skin elasticity)

1.4 Severe Ophthalmic Impairment

Seven patients from the present study (12%) sustained unilateral central vision loss and 8 patients (14%) bilateral central vision loss. Mean area occupied by calcification was 19.24%

in patients with vision loss in at least one eye, and 18.54% in patients with bilateral vision loss. Difference in dermal calcification area was not statistically significant (p=0.35 for vision

(25)

12 1.5 Cardiovascular Manifestations

Seven patients (11.8%) presented with severe cardiovascular disease defined as coronary artery disease with a history of myocardial infarction or angioplasty and/or a stroke (or transient ischemic attack) and/or revascularized disease of the lower limb arteries. Mean area occupied by calcification in these patients was 12.57% whereas it reached 16.72% in patients who did not suffer from severe cardiovascular disease. No statistically significant difference was observed between the two groups (p=0.17).

1.6 Coronary Calcium Scoring

Coronary calcium scores were available for 39 patients. The mean score was 192 and the median score was 5 (SD=531). No link was found between area occupied by calcification and coronary calcium scores (Spearman ρ=-0.007, p=0.97) (Figure 6).

Figure 6: Percentage area occupied by calcification in terms of coronary calcium scores.

1.7 Lower Limb Arterial Calcification (LLAC) Scores

Coronary calcium scores were available for 53 patients. The mean score was 3.2 and the median score was 0.69 (SD=6.7). No link was observed between area occupied by calcification and LLAC scores (Spearman ρ=0.003, p=0.98) (Figure 7).

(26)

Figure 7: Percentage area occupied by calcification in terms of LLAC scores

1.8 Plasma PPi

Plasma PPi levels were determined in 36 patients. No statistically significant link was observed between plasma PPi levels and area occupied by calcification (Spearman ρ=0.21, p=0.23) (Figure 8).

Figure 8. Percentage area occupied by calcification in terms of PPi levels

(27)

14

2. Area Occupied by Elastic Fibers

Mean percentage area occupied by fragmented elastic fibers was 14.19% (SD=6.0). The distribution of the areas occupied by elastic fibers did not follow a normal distribution pattern.

No link was found between area occupied by calcification and area occupied by elastic fibers (Spearman ρ= 0.11, p= 0.42) (Figure 9).

Figure 9: Area occupied by elastic fibers in terms of area occupied by calcification

2.1 Age

No link was found between age and area occupied by elastic fibers (Spearman ρ=-0.11, p=0.39) (Figure 10).

(28)

Figure 10: Area occupied by elastic fibers in terms of age

2.2 Sex

There was no statistically significant difference in area occupied by elastic fibers between men and women (p=0.32).

2.3 Skin Involvement

No link was found between number of affected cutaneous sites and area occupied by elastic fibers (Spearman ρ=0.15, p=0.25) (Figures 4 and 11).

Figure 11: Area occupied by elastic fibers in terms of number of affected skin sites

(29)

16 No statistically significant difference in severity of skin involvement was found between the medians (p=0.56) (Figure 12).

Figure 12: Box plots. Area occupied by elastic fibers in terms of severity of skin involvement.

2.4 Severe Ophthalmic Impairment

Mean area occupied by fragmented elastic fibers was 12.51% in patients with vision loss in at least one eye and 12.19% in patients with bilateral vision loss. There was no statistically significant difference in area occupied by elastic fibers (p=0.26 for vision loss in at least one eye and p=0.47 for bilateral vision loss).

2.5 Cardiovascular Manifestations

Mean area occupied by elastic fibers in patients with severe cardiovascular disease was 14.04% whereas it was 14.19% in patients who were not affected by severe cardiovascular disease. No statistically significant difference was found between the two groups (p=0.82).

2.6 Coronary Calcium Scoring

No linear association was observed between area occupied by elastic fibers and coronary calcium scores (Spearman ρ=-0.35, p=0.03) (Figure 13).

(30)

Figure 13: Area occupied by elastic fibers in terms of coronary calcium scores

2.7 Lower Limb Arterial Calcification (LLAC) Scores

No link was found between area occupied by elastic fibers and LLAC scores (Spearman ρ= - 0.22, p=0.11) (Figure 14).

Figure 14: Area occupied by elastic fibers in terms of LLAC scores

2.8 Plasma PPi

No link was found between plasma PPi levels and area occupied by elastic fibers (Spearman ρ= -0.13, p=0.45) (Figure 15).

(31)

18 Figure 15: Area occupied by elastic fibers in terms of PPi ratios (patient/control).

(32)

DISCUSSION

The intensity of histological abnormalities is highly variable in PXE skin biopsies.

Severity and extent of skin involvement are in the present study associated with area occupied by calcification in skin biopsies. No link was however found between age, severe cardiovascular disease, vision loss and biopsy calcification rates. There was no evidence of correlation between area occupied by elastic fibers and any of the parameters investigated.

Little data is available in the literature on the methods used to calculate skin biopsy calcification rates. In several papers (32,33), skin biopsies were graded by a blinded dermatopathologist according to density of Von Kossa staining, and any alterations to the degree of elastic fiber calcification were assessed using von Kossa staining per unit area of dermis. In a cohort of 40 patients (32), quantification of calcification using a similar method to that employed in the present study found a mean calcified area of 29.52. Any deviation from the mean value found in our work can be partly explained by magnification (x20 versus x10 in our study), choice of site, and discrepancy between assessment by software and by a dermatopathologist.

A number of studies address area occupied by elastic fibers. Uitto et al. (34) found that elastic fibers occupied 2.1 +/- 1.1% of the dermis in the skin of 10 healthy control subjects.

Volume fractions occupied by elastic fibers in PXE lesions increased as much as 6-fold, whereas values in unaffected skin areas in the same patients were within normal limits. This procedure, as in our study, involved examining the elastic fibers through a camera unit attached to a microscope. The black and white images sensing various grey levels were converted to binary images after a threshold had been selected. Data analysis was conducted using a computer program designed to render the properties of the images obtained.

(33)

20 Contrary to our findings, Gogly et al. (35) reported good correlation between area fraction occupied by elastic fibers in unexposed skin (inner aspect of upper arm) and aging. In sun- protected skin tissue, significant correlation was found between area fraction occupied by elastic fibers in the dermis and in the deep temporal artery. A mere 16 patients were included in this study. Our results most probably differ because they are based on pathological cases.

According to our data, the rate of skin calcification is independent of age at time of biopsy, highlighting the relevance of this parameter for investigating PXE pathophysiology.

Our findings regarding a link between areas occupied by elastic fibers and by calcification were surprising. We expected to find a strong association since PXE is characterized by calcification of elastic fibers in connective tissues.

The reason for such findings may conceivably lie in the subjectivity of the method used to measure area occupied by elastic fibers (Appendix III). Moreover, reproducibility of the 3 measurements taken to calculate the area occupied by elastic fibers was poor: mean variation in each measurement was 4.7.

Kranenbourg (28) reported that 33% of PXE patients had a history of peripheral artery disease, 6% of coronary disease and 9% of cerebral vascular disease. This appears consistent with the percentage of patients from our cohort (11.8%) with severe cardiovascular disease.

Given that PXE is a disease that affects blood vessels, correlation between calcification rates and vascular damage might be expected. However, area occupied by calcification is apparently no more substantial in patients who have sustained serious cardiovascular events.

Inaccurate scoring when assessing severe cardiovascular events (0 or 1) may account for this finding. It would be interesting to differentiate between the various types of cardiovascular injury and to rank lesion intensity. In our opinion, systolic pressure index is

(34)

inappropriate since it is regarded as pathological both when it is below 0.9 and above 1.2.

Furthermore, calcium scores would equally appear to be unconnected with area occupied by elastic fibers.

Risseeuw (36) reports that in the population of patients aged 50 years and over, 36.9% had some form of visual impairment, and 15.4% were legally blind. Patient proportions increased with age: in their sixth decade, 21.1% of all patients were visually impaired. This percentage increased in the seventh decade to 43.6% and increased further to 85.7% in the eighth decade. Additionally, the percentage of legally blind individuals increased from 3.4% in patients in their fifth decade to 42.9% in patients in their eighth decade. In our study, 10%

had bilateral vision loss. The mean age of these patients was 60.2 years. 25% of patients 50 years and over were found to have bilateral vision loss in the present study.

In France, legal blindness is defined as better eye visual acuity of less than 1/20 after best correction. In most Anglo-Saxon countries, legal blindness is defined as better eye visual acuity of less than or equal to 1/10. It is therefore difficult to compare data. We were unable to access the findings from every ophthalmic examination. It would be of interest to discover the proportion of patients treated with intraocular anti-VEGF as this treatment is potentially influential in preventing vision loss.

Navasiolava et al. (30) found a statistically significant link between number of affected skin areas, age, sex and development of complications. Number of affected skin areas was significantly higher in patients with severe cardiovascular disease, bilateral vision loss, and vision loss in at least one eye.

(35)

22 Utani et al. (31) demonstrated in a small cohort that there was a link between higher numbers of affected skin sites and both cardiovascular disease and width of angioid streaks.

Furthermore, mucosal lesions were associated with cardiovascular events.

We observed a link between area occupied by calcification and number of affected skin sites, but we found no connection with cardiovascular disease or ophthalmic impairment. These findings may result from our small patient sample size.

One obvious limitation to our work is that our trial was conducted at a single point in time.

We were only able to include 59 patients which, despite representing a large cohort given the rarity of PXE, is too small a cohort to yield any significant results.

Possible reasons for measurement inaccuracy are age of slides, quality of staining or cut, choice of area to be photographed and resolution of data processing.

A further study limitation is its lack of reproducibility. There is no guarantee that a biopsy taken directly adjacent to a previous site will detect a similar rate of calcification or fragmented elastic fibers.

The present work enriches current data on the natural course of PXE. We have shown that calcification rates are highly variable in PXE patients. However skin biopsy is not an optimal method for detecting cardiovascular or ophthalmic damage.

ImageJ facilitates assessment of abnormalities and could conceivably be used as a form of therapeutic monitoring. It would be beneficial to assess the calcification rate of a given skin site over the long term using a non-invasive method such as non-linear optical microscopy (37).

(36)

CONCLUSION

The intensity of histological abnormalities in skin biopsies obtained from PXE patients is highly variable. Calcification is correlated with the severity and extent of skin involvement, but not with severe cardiovascular disease or vision loss. In practical terms, severe skin damage in a PXE patient invariably indicates cardiovascular or ophthalmic complications to a dermatologist. The intensity of histological lesions is not predictive of such complications. It is therefore essential to ensure that regular checkups are carried out to detect any PXE complications.

(37)

24

REFERENCES

1. Chassaing N. Pseudoxanthoma elasticum: a clinical, pathophysiological and genetic update including 11 novel ABCC6 mutations. J Med Genet. Apr 2005;42(12):881-92.

2. Favre G, Laurain A, Aranyi T, Szeri F, Fulop K, Le Saux O, et al. The ABCC6 Transporter:

A New Player in Biomineralization. Int J Mol Sci. Sep 2017;18(9):1941.

3. Germain DP. Pseudoxanthoma elasticum. Orphanet J Rare Dis. Dec 2017;12(1).

4. Marconi B, Bobyr I, Campanati A, Molinelli E, Consales V, Brisigotti V, et al.

Pseudoxanthoma elasticum and skin: Clinical manifestations, histopathology, pathomechanism, perspectives of treatment. Intractable Rare Dis Res. Aug 2015;4(3):113-22.

5. Uitto J, Jiang Q, Váradi A, Bercovitch LG, Terry SF. Pseudoxanthoma Elasticum:

Diagnosis Features, Classification, and Treatment Options. Expert Opin Orphan Drugs.

Jun 2014;2(6):567-77.

6. Pipelart V, Leruez S, Martin L, Navasiolava N, Henni S, Ebran J-M. Study of fundus examination by age in 158 pseudoxanthoma elasticum patients. J Fr Ophtalmol. Sep 2018;41(7):592-602.

7. Georgalas I, Papaconstantinou D, Koutsandrea C, Kalantzis G, Karagiannis D, Georgopoulos G, et al. Angioid streaks, clinical course, complications, and current therapeutic management. Ther Clin Risk Manag. 2009;5:81-9.

8. Kauw F, Kranenburg G, Kappelle LJ, Hendrikse J, Koek HL, Visseren FLJ, et al. Cerebral disease in a nationwide Dutch pseudoxanthoma elasticum cohort with a systematic review of the literature. J Neurol Sci. Feb 2017;373:167-72.

9. Kranenburg G, de Jong PA, Mali WP, Attrach M, Visseren FLJ, Spiering W. Prevalence and severity of arterial calcifications in pseudoxanthoma elasticum (PXE) compared to hospital controls. Novel insights into the vascular phenotype of PXE. Atherosclerosis.

2017;256:7-14.

10. Lefthériotis G, Omarjee L, Saux OL, Henrion D, Abraham P, Prunier F, et al. The vascular phenotype in Pseudoxanthoma elasticum and related disorders: contribution of a genetic disease to the understanding of vascular calcification. Front Genet. Feb 2013;4.

11. Letavernier E, Bouderlique E, Zaworski J, Martin L, Daudon M. Pseudoxanthoma Elasticum, Kidney Stones and Pyrophosphate: From a Rare Disease to Urolithiasis and Vascular Calcifications. Int J Mol Sci. Dec 2019;20(24).

12. Hosen MJ, Lamoen A, De Paepe A, Vanakker OM. Histopathology of Pseudoxanthoma Elasticum and Related Disorders: Histological Hallmarks and Diagnostic Clues.

Scientifica. 2012.

(38)

13. Lebwohl M, Phelps RG, Yannuzzi L, Chang S, Schwartz I, Fuchs W. Diagnosis of pseudoxanthoma elasticum by scar biopsy in patients without characteristic skin lesions.

N Engl J Med. Aug 1987;317(6):347-50.

14. Berthin C, Phan A, Navasiolava N, Michalak S, Humeau H, Grimaux X, et al.

Dermoscopic phenotype of pseudoxanthoma elasticum skin lesions: a study of 16 patients. J Eur Acad Dermatol Venereol. Mar 2019

15. Persechino F, Giordano D, Marini CD, Franceschini C, Ardigò M, Persechino S.

Dermoscopy, Optical Coherence Tomography, and Histological Correlation of Pseudoxanthoma Elasticum. Dermatol Pract Concept. Jul 2019;209-10.

16. Vos A, Kranenburg G, de Jong PA, Mali WPTM, Van Hecke W, Bleys RLAW, et al. The amount of calcifications in pseudoxanthoma elasticum patients is underestimated in computed tomographic imaging; a post-mortem correlation of histological and computed tomographic findings in two cases. Insights Imaging. Aug 2018;9(4):493-8.

17. Jansen RS, Kucukosmanoglu A, de Haas M, Sapthu S, Otero JA, Hegman IEM, et al.

ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release. Proc Natl Acad Sci. Dec 2013;110(50):20206-11.

18. Jansen RS, Duijst S, Mahakena S, Sommer D, Szeri F, Váradi A, et al. ABCC6–Mediated ATP Secretion by the Liver Is the Main Source of the Mineralization Inhibitor Inorganic Pyrophosphate in the Systemic Circulation—Brief Report. Arterioscler Thromb Vasc Biol.

Sep 2014;34(9):1985-9.

19. Pomozi V, Brampton C, van de Wetering K, Zoll J, Calio B, Pham K, et al. Pyrophosphate Supplementation Prevents Chronic and Acute Calcification in ABCC6-Deficient Mice. Am J Pathol. Jun 2017;187(6):1258-72.

20. Lomashvili KA, Narisawa S, Millán JL, O’Neill WC. Vascular calcification is dependent on plasma levels of pyrophosphate. Kidney Int. Jun 2014;85(6):1351-6.

21. Zhao J, Kingman J, Sundberg JP, Uitto J, Li Q. Plasma PPi Deficiency Is the Major, but Not the Exclusive, Cause of Ectopic Mineralization in an Abcc6 Mouse Model of PXE. J Invest Dermatol. Nov 2017;137(11):2336-43.

22. Carrillo-Linares JL, García-Fernández MI, Morillo MJ, Sánchez P, Rioja J, Barón FJ, et al.

The Effects of Parenteral K1 Administration in Pseudoxanthoma Elasticum Patients Versus Controls. A Pilot Study. Front Med. Apr 2018;5:86.

23. Gorgels TGMF, Waarsing JH, Herfs M, Versteeg D, Schoensiegel F, Sato T, et al. Vitamin K supplementation increases vitamin K tissue levels but fails to counteract ectopic calcification in a mouse model for pseudoxanthoma elasticum. J Mol Med Berl Ger. Nov 2011;89(11):1125-35.

24. Vanakker OM, Martin L, Schurgers LJ, Quaglino D, Costrop L, Vermeer C, et al. Low serum vitamin K in PXE results in defective carboxylation of mineralization inhibitors similar to the GGCX mutations in the PXE-like syndrome. Lab Invest. Jun 2010;90(6):895-905.

(39)

26 25. Li Q, Arányi T, Váradi A, Terry SF, Uitto J. Research Progress in Pseudoxanthoma Elasticum and Related Ectopic Mineralization Disorders. J Invest Dermatol. Mar 2016;136(3):550-6.

26. Dedinszki D, Szeri F, Kozák E, Pomozi V, Tőkési N, Mezei TR, et al. Oral administration of pyrophosphate inhibits connective tissue calcification. EMBO Mol Med.

2017;9(11):1463-70.

27. Pomozi V, Julian CB, Zoll J, Pham K, Kuo S, Tőkési N, et al. Dietary Pyrophosphate Modulates Calcification in a Mouse Model of Pseudoxanthoma Elasticum: Implication for Treatment of Patients. J Invest Dermatol. May 2019;139(5):1082-8.

28. Kranenburg G, de Jong PA, Bartstra JW, Lagerweij SJ, Lam MG, Ossewaarde-van Norel J, et al. Etidronate for Prevention of Ectopic Mineralization in Patients With Pseudoxanthoma Elasticum. J Am Coll Cardiol. Mar 2018;71(10):1117-26.

29. Bartstra JW, de Jong PA, Kranenburg G, Wolterink JM, Isgum I, Wijsman A, et al.

Etidronate halts systemic arterial calcification in pseudoxanthoma elasticum.

Atherosclerosis. Jan 2020;292:37-41.

30. Navasiolava N, Gnanou M, Douillard M, Saulnier P, Aranyi T, Ebran J-M, et al. The extent of pseudoxanthoma elasticum skin changes is related to cardiovascular complications and visual loss: a cross-sectional study. Br J Dermatol. 2019;180(1):207-8.

31. Utani A, Tanioka M, Yamamoto Y, Taki R, Araki E, Tamura H, et al. Relationship between the distribution of pseudoxanthoma elasticum skin and mucous membrane lesions and cardiovascular involvement. J Dermatol. Feb 2010;37(2):130-6.

32. Rose S, On SJ, Fuchs W, Chen C, Phelps R, Kornreich D, et al. Magnesium supplementation in the treatment of pseudoxanthoma elasticum: A randomized trial. J Am Acad Dermatol. Jul 2019;81(1):263-5.

33. Yoo JY, Blum RR, Singer GK, Stern DK, Emanuel PO, Fuchs W, et al. A randomized controlled trial of oral phosphate binders in the treatment of pseudoxanthoma elasticum. J Am Acad Dermatol. Aug 2011;65(2):341-8.

34. Uitto J, Paul JL, Brockley K, Pearce RH, Clark JG. Elastic fibers in human skin:

quantitation of elastic fibers by computerized digital image analyses and determination of elastin by radioimmunoassay of desmosine. Lab Investig J Tech Methods Pathol. Oct 1983;49(4):499-505.

35. Gogly B, Godeau G, Septier D, Hornebeck W, Pellat B, Jeandel C. Measurement of the amounts of elastic fibers in the skin and temporal arteries of healthy aged individuals by automated image analysis. Gerontology. 1998;44(6):318-23.

36. Risseeuw S, Ossewaarde-van Norel J, Klaver CCW, Colijn JM, Imhof SM, van Leeuwen R.

Visual Acuity in Pseudoxanthoma Elasticum. Retina Phila Pa. Aug 2019;39(8):1580-7.

37. Kiss N, Fésűs L, Bozsányi S, Szeri F, Van Gils M, Szabó V, et al. Nonlinear optical microscopy is a novel tool for the analysis of cutaneous alterations in pseudoxanthoma elasticum. Lasers Med Sci. May 2020

(40)

LIST OF FIGURES

Figure 1: Flow Chart 8

Figure 2: Area occupied by calcification in terms of age 9

Figure 3: Positive correlation between area occupied by calcification and number of affected skin sites 10

Figure 4: ROC curve for number of affected skin sites ≥9 10

Figure 5: Box plots. Percentage area occupied by calcification in terms of severity of skin involvement 11

Figure 6: Percentage area occupied by calcification in terms of coronary calcium scores 12

Figure 7: Percentage area occupied by calcification in terms of LLAC scores 13

Figure 8: Percentage area occupied by calcification in terms of PPi levels 13

Figure 9: Area occupied by elastic fibers in terms of area occupied by calcification 14

Figure 10: Area occupied by elastic fibers in terms of age 15

Figure 11: Area occupied by elastic fibers according to number of affected skin sites 15

Figure 12: Box plots. Percentage area occupied by elastic fibers in terms of severity of skin involvement 16

Figure 13: Area occupied by elastic fibers in terms of coronary calcium scores 17

Figure 14: Area occupied by elastic fibers in terms of LLAC scores 17

Figure 15: Area occupied by elastic fibers in terms of PPi ratios (patient/control) 18

(41)

28

APPENDICES

Appendix I: Procedure for Calculating Area Occupied by Calcification Using ImageJ Software

The procedure used to calculate the area occupied by calcification using ImageJ software is as follows:

1- File/Open: Image in JPEG format. [Image A]: histology X10 of antecubital biopsy using von Kossa staining

2- [Image B] Image/ type/ 8-bit

3- Insert scale for measurement: *straight* draw a line on the image scale 4- Analyze/set scale: record scale size in “distance in pixels”

5- [Image C] Process/ Filters/ Median: we arbitrarily selected 4 in Radius to avoid overestimation

6- [Image D] Process/ Binary/ Make binary

7- [Image E] *rectangle* draw a square with sides of 700µm

8- Analyze/ Set measurements/ Area fraction (deselect other options)

9- Place the square on the area of interest (the most calcified)/ Analyze/ Measure

(43)

II Appendix II: Procedure for Calculating Area Occupied by Elastic Fibers Using

ImageJ software

1- File/Open: Image in JPEG format. [Image A]: histology X10 of antecubital biopsy with Verhoeff-van-Gieson staining.

2- Insert scale for measurement: *straight* draw a line on the image scale 3- Analyze/ set scale: record the scale size in “distance in pixels”

4- *rectangle* draw a square with sides of 700µm 5- [Image B] Image/ Crop

6- [Image C] Image/ type/ 8-bit

7- [Image D] Image/ Adjust/ Threshold/ Move the cursor if the software is incorrectly estimated/ Apply

8- [Image E] Analyze/ Analyze particle/ Size (pixel^2): 1-infinity, Circularity 0-1/ Ok.

The area occupied by elastic fibers in this image is estimated at 27.19%.

(44)

Appendix III: Subjectivity of Method Used to Measure Elastic Fibers

A: Verhoeff staining with elastic fibers in black.

B: Percentage estimated at 31.82%.

C: Percentage for same slide estimated at 19.27%.

(45)

(46)

DELAITRE Léa

Recherche d’une association entre la sévérité de l’atteinte clinique et l’intensité des lésions histologiques dermiques chez les patients atteints d’un pseudoxanthome

élastique

Mots-clés: pseudoxanthome élastique, calcifications, fibres élastiques

Association between Severity of Clinical Impairment and Intensity of Dermal Histological Lesions in Pseudoxanthoma Elasticum

Keywords: pseudoxanthoma elasticum, calcification, elastic fibers

AB ST RACT

Introduction: Pseudoxanthoma elasticum (PXE) is a rare, systemic metabolic disease with autosomal recessive inheritance (OMIM 264800) caused by ABCC6 gene mutation in the majority of cases. PXE is characterized by calcification and fragmentation of elastic fibers (or elastorrhexis) mainly affecting the skin, retina and artery walls. The severity of skin, cardiovascular, ophthalmic and histological damage varies from one subject to another. At present there is no basis (in terms of histology for instance) for predicting the clinical course of organ damage in a given patient. Our objective was to find a correlation between intensity of histological PXE lesions and severity of organ damage.

Methods: All patients followed at our specialist center with indisputable diagnosis of PXE who underwent elbow fold biopsy between 2008 and 2019 were included. A 700x700µm area was selected on each histological slide and the percentage area occupied by calcification and elastic fibers was calculated using ImageJ software. The severity of the various clinical diseases was assessed qualitatively.

Results: 59 patients were included. Mean percentage area occupied by calcification was 16.43% (SD=11.8).

Mean percentage area occupied by elastic fibers was 14.19% (SD=6.0). Area occupied by calcification demonstrated moderate correlation with number of affected skin sites (Spearman ρ=0.35, p<0.01) and with severity of cutaneous damage at biopsied sites (Spearman ρ=0.47, p<0.01). There was no correlation between area occupied by calcification and age, plasma PPi levels (Spearman ρ=0.21, p=0.23), severe cardiovascular manifestations and vision loss. There was no evidence of correlation between area occupied by dystrophic elastic fibers and any of the parameters investigated.

Conclusion: The intensity of histological abnormalities in PXE patients is highly variable. Calcification was found to correlate with severity and extent of skin involvement alone. In practical terms, severe skin damage in a PXE patient indicates cardiovascular or ophthalmic complications to a dermatologist. The intensity of histological lesions is not predictive of such complications. It is therefore essential to ensure that regular checkups are carried out to detect any PXE complications.

RÉSUMÉ

Introduction : Le pseudoxanthome élastique (PXE) est une maladie métabolique systémique, rare, autosomique récessive, due à des mutations du gène ABCC6. Le PXE est caractérisé par une calcification et une fragmentation des fibres élastiques (élastorrhexie) affectant principalement la peau, la rétine et les parois artérielles. La sévérité de l’atteinte cutanée, cardiovasculaire et ophtalmologique varie d’un sujet à l’autre, et nous ne disposons pas aujourd’hui d’éléments, par exemple histologiques, permettant de prédire l’évolutivité des atteintes organiques chez un patient donné. Notre objectif était de trouver une corrélation entre l’intensité des lésions histologiques de PXE et la sévérité des atteintes d’organes.

Méthodes : Tous les patients suivis au centre de référence, avec un diagnostic certain de PXE et ayant eu une biopsie au pli du coude entre 2008 et 2019 ont été inclus. Nous avons sélectionné une zone de 700x700µm sur chaque lame histologique et mesuré le pourcentage de l’aire occupée par les calcifications et par les fibres élastiques à l’aide du logiciel ImageJ. La sévérité des différentes atteintes cliniques était appréciée de façon qualitative.

Résultats : 59 patients ont été inclus. Le pourcentage moyen de l’aire occupée par les calcifications était de 16,43% (SD=11,8). L’aire occupée par les fibres élastiques dystrophiques était de 14,19% (SD=6,0). L’aire occupée par les calcifications était modérément corrélée au nombre de sites cutanés atteints (Spearman ρ=0,35, p<0,01) et à la sévérité de l’atteinte cutanée au site biopsié (Spearman ρ=0,47, p<0,01). Il n’y avait pas de corrélation entre l’aire occupée par les calcifications et l’âge, le taux de PPi plasmatique (Spearman ρ=0,21, p=0,23), l’existence de manifestations cardiovasculaires sévères et d’une cécité. L’aire occupée par les fibres élastiques dystrophiques n’était corrélée avec aucun des paramètres étudiés.

Conclusion : L’intensité des anomalies histologiques chez les patients PXE est très variable. Les calcifications ne sont corrélées qu’avec la sévérité et l’étendue de l’atteinte cutanée. En pratique, pour un dermatologue, l’atteinte cutanée sévère chez un patient PXE est associée aux complications cardiovasculaires ou ophtalmologiques.

L’intensité des lésions histologiques ne permet pas de prédire ces complications. Il est donc nécessaire de réaliser des bilans réguliers afin de dépister les complications du PXE.