Amelogenesis imperfecta (AI) is a heterogeneous group of genetic rare diseases disrupting enamel development (Smith et al., Front Physiol, 2017a, 8, 333). The clinical enamel phenotypes can be described as hypoplastic, hypomineralized or hypomature and serve as a basis, together with the mode of inheritance, to Witkop's classification (Witkop, J Oral Pathol, 1988, 17, 547-553). AI can be described in isolation or associated with others symptoms in syndromes. Its occurrence was estimated to range from 1/700 to 1/14,000. More than 70 genes have currently been identified as causative. Objectives: We analyzed using next-generation sequencing (NGS) a heterogeneous cohort of AI patients in order to determine the molecular etiology of AI and to improve diagnosis and disease management. Methods: Individuals presenting with so called "isolated" or syndromic AI were enrolled and examined at the Reference Centre for Rare Oral and Dental Diseases (O-Rares) using D4/phenodent protocol (www.phenodent.org). Families gave written informed consents for both phenotyping and molecular analysis and diagnosis using a dedicated NGS panel named GenoDENT. This panel explores currently simultaneously 567 genes. The study is registered under NCT01746121 and NCT02397824 (https://clinicaltrials.gov/). Results: GenoDENT obtained a 60% diagnostic rate. We reported genetics results for 221 persons divided between 115 AI index cases and their 106 associated relatives from a total of 111 families. From this index cohort, 73% were diagnosed with non-syndromic amelogenesis imperfecta and 27% with syndromic amelogenesis imperfecta. Each individual was classified according to the AI phenotype. Type I hypoplastic AI represented 61 individuals (53%), Type II hypomature AI affected 31 individuals (27%), Type III hypomineralized AI was diagnosed in 18 individuals (16%) and Type IV hypoplastic-hypomature AI with taurodontism concerned 5 individuals (4%). We validated the genetic diagnosis, with class 4 (likely pathogenic) or class 5 (pathogenic) variants, for 81% of the cohort, and identified candidate variants (variant of uncertain significance or VUS) for 19% of index cases. Among the 151 sequenced variants, 47 are newly reported and classified as class 4 or 5. The most frequently discovered genotypes were associated with MMP20 and FAM83H for isolated AI. FAM20A and LTBP3 genes were the most frequent genes identified for syndromic AI. Patients negative to the panel were resolved with exome sequencing elucidating for example the gene involved ie ACP4 or digenic inheritance. Conclusion: NGS GenoDENT panel is a validated and cost-efficient technique offering new perspectives to understand underlying molecular mechanisms of AI. Discovering variants in genes involved in syndromic AI (CNNM4, WDR72, FAM20A ... ) transformed patient overall care. Unravelling the genetic basis of AI sheds light on Witkop's AI classification.

APHM Hôpitaux Universitaires de Marseille Hôpital Timone Competence Center for Rare Oral and Dental Diseases Marseille France

Centre Hospitalier Régional Universitaire de Nancy Université de Lorraine Competence Center for Rare Oral and Dental Diseases Nancy France

Centre Hospitalier Universitaire de Lyon Competence Center for Rare Oral and Dental Diseases Lyon France

Centre Hospitalier Universitaire de Nantes Competence Center for Rare Oral and Dental Diseases Nantes France

Centre Hospitalier Universitaire de Nice Competence Center for Rare Oral and Dental Diseases Nice France

Centre Hospitalier Universitaire Rangueil Toulouse Competence Center for Rare Oral and Den tal Diseases Toulouse France

Department of Stomatology Charles University 2nd Faculty of Medicine and Motol University Hospital Prague Czechia

Eastman Dental Institute University College London London United Kingdom

Faculty of Dentistry International University of Rabat CReSS Centre de recherche en Sciences de la Santé Rabat Morocco

Faculty of Dentistry Khon Kaen University Khon Kaen Thailand

Hôpitaux Universitaires de Strasbourg Laboratoires de diagnostic génétique Institut de Génétique Médicale d'Alsace Strasbourg France

Hôpitaux Universitaires de Strasbourg Pôle de Médecine et Chirurgie Bucco dentaires Hôpital Civil Centre de référence des maladies rares orales et dentaires O Rares Filiére Santé Maladies rares TETE COU European Reference Network ERN CRANIO Strasbourg France

Instituto de Investigación en Ciencias Odontológicas Facultad de Odontología Universidad de Chile Santiago Chile

Rothschild Hospital Public Assistance Paris Hospitals Paris France

Unité de génétique médicale et d'oncogénétique CHU Hassan 2 Fes Morocco

Université de Strasbourg Faculté de Chirurgie Dentaire Strasbourg France

Université de Strasbourg Institut d'études avancées Strasbourg France

Université de Strasbourg Institut de Génétique et de Biologie Moléculaire et Cellulaire IN SERM U1258 CNRS UMR7104 Illkirch France

Université de Strasbourg Laboratoire de Biomatériaux et Bioingénierie Inserm UMR_S 1121 Strasbourg France

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