Feliciana Mariotti ^1* Anna Pira ¹ Naomi DE LUCA ¹ Anna Rita Giampetruzzi ² Filomena Russo ² Amilcare Cerri ³ Giulia Gasparini ⁴ Emanuele Cozzani ⁴ Angelo Valerio Marzano ⁵ Emiliano Antiga ⁶ Marzia Caproni ⁷ Pietro Quaglino ⁸ Marco Carrozzo ⁹ Biagio Didona ¹⁰ Giovanni Di Zenzo ¹

• ¹ Molecular and Cell Biology Laboratory, Istituto Dermopatico dell’Immacolata (IDI)-IRCCS, Rome, Italy
• ² Dermatology Unit, Istituto Dermopatico dell'Immacolata (IDI)-IRCCS, Rome, Italy
• ³ Dermatological Clinic, Department of Health Sciences, University of Milan, AO Santi Paolo e Carlo, Milan, Italy
• ⁴ Dermatology Unit, IRCCS Ospedale Policlinico San Martino,, Section of Dermatology, Department of Health Sciences (DISSAL), University of Genoa, Genoa, Italy
• ⁵ Department of Pathophysiology and Transplantation, Università Degli Studi di Milano, Dermatology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Lombardy, Italy
• ⁶ Section of Dermatology, Department of Health Sciences, University of Florence, Florence, Italy
• ⁷ Immunopathology and Rare Skin Diseases Unit, Section of Dermatology, Department of Health Sciences, Azienda Unità Sanitaria Locale Toscana Centro, ERN-Skin member, University of Florence, Florence, Italy
• ⁸ Dermatologic Clinic, Department of Medical Sciences, University of Turin, Turin, Italy
• ⁹ Oral Medicine Department, School of Dental Sciences, Newcastle University, Newcastle upon Tyne, United Kingdom
• ¹⁰ Rare Diseases Unit, Istituto Dermopatico dell’Immacolata (IDI)-IRCCS, Rome, Italy

Background: Bullous pemphigoid (BP) and mucous membrane pemphigoid (MMP) are rare autoimmune blistering disorders characterized by autoantibodies (autoAbs) targeting dermo-epidermal junction components such as BP180 and BP230. The differential diagnosis, based on both the time of appearance and the extension of cutaneous and/or mucosal lesions, is crucial to distinguish these diseases for improving therapy outcomes and delineating the correct prognosis, however, in some cases it can be challenging. In addition, negative results obtained by commercially available ELISAs with BP and MMP sera, especially from patients with ocular involvement, often delay diagnosis and treatment, leading to a greater risk of poor outcomes. Objectives: Our aim was to find potentially different reactivity profiles in BP and MMP and improve available approaches for diagnosis with focus on ocular MMP. Methods: Two cohorts of 90 BP and 90 MMP, recruited in different Italian clinical centers, were characterized also employing a novel ELISA based on BP180 extracellular domain (ECD-BP180). Results: IgG reactivity to BP180 and BP230 in MMP sera was significantly reduced in comparison with BP, mostly affecting BP230 and E-1080 (53% and 36% in BP vs 11% and 3% in MMP, respectively, p<0.0001). The combined sensitivity of BP180-NC16A- and ECD-BP180-ELISAs was greater compared to BP180-NC16A- and BP230-ELISAs both in BP (97% and 92%, respectively) and in MMP (42% and 31%, respectively). The present study shows that MMP patients with ocular involvement rarely reacted to BP180 by IgG in contrast with patients with oral and/or cutaneous involvement (p=0.0245 and p=0.0377, respectively), suggesting that an oral and/or cutaneous MMP positive to BP180 hardly evolves to ocular MMP. Of note, one-third of ocular MMP showed IgA reactivity to ECD-BP180 by immunoblotting. Conclusions: The present study provides several hints to perform a correct and timely diagnosis in BP and MMP, that is crucial for improving therapy outcomes and delineating the correct prognosis.