JMG Online First, published on October 26, 2015 as 10.1136/jmedgenet-2015-103302 Methods J Med Genet: first published as 10.1136/jmedgenet-2015-103302 on 26 October 2015. Downloaded from ORIGINAL ARTICLE A targeted next-generation sequencing assay for the molecular diagnosis of genetic disorders with orodental involvement Megana K Prasad,1 Véronique Geoffroy,1 Serge Vicaire,2 Bernard Jost,2 Michael Dumas,2 Stéphanie Le Gras,2 Marzena Switala,3,4 Barbara Gasse,5 Virginie Laugel-Haushalter,6 Marie Paschaki,1,6 Bruno Leheup,7 Dominique Droz,8 Amelie Dalstein,8 Adeline Loing,3 Bruno Grollemund,4 Michèle Muller-Bolla,9,10 Séréna Lopez-Cazaux,11 Maryline Minoux,3,4 Sophie Jung,3,4 Frédéric Obry,3,4 Vincent Vogt,3,4 Jean-Luc Davideau,4 Tiphaine Davit-Beal,5,12 Anne-Sophie Kaiser,13 Ute Moog,13 Béatrice Richard,14 Jean-Jacques Morrier,14 Jean-Pierre Duprez,14 Sylvie Odent,15 Isabelle Bailleul-Forestier,16 Monique Marie Rousset,17 Laure Merametdijan,18 Annick Toutain,19 Clara Joseph,20 Fabienne Giuliano,21 Jean-Christophe Dahlet,3 Aymeric Courval,22 Mustapha El Alloussi,23 Samir Laouina,23 Sylvie Soskin,24 Nathalie Guffon,25 Anne Dieux,26 Bérénice Doray,27 Stephanie Feierabend,28 Emmanuelle Ginglinger,29 Benjamin Fournier,30,31 Muriel de la Dure Molla,30,31 Yves Alembik,27 Corinne Tardieu,32 François Clauss,3,4 Ariane Berdal,30,31 Corinne Stoetzel,1 Marie Cécile Manière,3,4 Hélène Dollfus,1,33 Agnès Bloch-Zupan3,4,6 ▸ Additional material is ABSTRACT variety of orodental diseases. Furthermore, our panel will published online only. To view Background Orodental diseases include several contribute to better understanding the contribution of please visit the journal online (http://dx.doi.org/10.1136/ clinically and genetically heterogeneous disorders that these genes to orodental disease. jmedgenet-2015-103302). can present in isolation or as part of a genetic Trial registration numbers NCT01746121 and syndrome. Due to the vast number of genes implicated NCT02397824. For numbered affiliations see in these disorders, establishing a molecular diagnosis end of article. http://jmg.bmj.com/ can be challenging. We aimed to develop a targeted Correspondence to next-generation sequencing (NGS) assay to diagnose INTRODUCTION Professor Agnès Bloch-Zupan, mutations and potentially identify novel genes mutated Orodental disorders encompass a number and Faculty of Dentistry , University of Strasbourg, in this group of disorders. variety of diseases that affect the teeth and oral 8 rue St Elisabeth, Methods We designed an NGS gene panel that targets cavity. Broadly, these disorders can be classified into Strasbourg 67000, France; 585 known and candidate genes in orodental disease. anomalies of tooth number, shape and size (eg, [email protected] We screened a cohort of 101 unrelated patients without hypo/oligo/ano-dontia (collectively selective tooth on September 28, 2021 by guest. Protected copyright. a molecular diagnosis referred to the Reference Centre Received 6 June 2015 agenesis (STHAG)), microdontia, globodontia), Revised 8 September 2015 for Oro-Dental Manifestations of Rare Diseases, anomalies of tooth structure (eg, amelogenesis Accepted 24 September 2015 Strasbourg, France, for a variety of orodental disorders imperfecta (AI), hereditary dentin disorders) and including isolated and syndromic amelogenesis anomalies of tooth eruption. The prevalence of imperfecta (AI), isolated and syndromic selective tooth these disorders varies from relatively common agenesis (STHAG), isolated and syndromic (4.2% for hypodontia in the Caucasian popula- dentinogenesis imperfecta, isolated dentin dysplasia, tion)1 to extremely rare (1 in 100 000 for dentin otodental dysplasia and primary failure of tooth dysplasia (DD) type 1).2 eruption. Orodental disorders can have a genetic, environ- Results We discovered 21 novel pathogenic variants mental or multifactorial basis.34Although evidence and identified the causative mutation in 39 unrelated demonstrates a role for environmental pollutants patients in known genes (overall diagnostic rate: 39%). such as dioxins and fluoride in developmental Among the largest subcohorts of patients with isolated enamel defects,56a number of studies have also AI (50 unrelated patients) and isolated STHAG (21 demonstrated a strong genetic aetiology for several To cite: Prasad MK, unrelated patients), we had a definitive diagnosis in 14 orodental diseases (reviewed in refs. 7–9). Among Geoffroy V, Vicaire S, et al. (27%) and 15 cases (71%), respectively. Surprisingly, the >5000 known genetic syndromes, >900 have J Med Genet Published 10 Online First: [please include COL17A1 mutations accounted for the majority of orodental/craniofacial features. Even in the case Day Month Year] autosomal-dominant AI cases. of isolated orodental diseases, significant genetic doi:10.1136/jmedgenet- Conclusions We have developed a novel targeted NGS heterogeneity exists, with several of the same genes 2015-103302 assay for the efficient molecular diagnosis of a wide being involved in isolated and syndromic forms of Prasad MK, et al. J Med Genet 2015;0:1–13. doi:10.1136/jmedgenet-2015-103302 1 Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence. Methods J Med Genet: first published as 10.1136/jmedgenet-2015-103302 on 26 October 2015. Downloaded from disease. For instance, mutations in eight genes have been impli- Patient phenotype was recorded using D[4]/Phenodent (http:// cated in STHAG (PAX9, MSX1, LTBP3, AXIN2, WNT10A, EDA, www.phenodent.org). – EDARADD and EDAR),11 17 of which several (MSX1, WNT10A, EDA, EDARADD and EDAR) have also been linked with forms 18–20 Gene selection and targeted capture design of ectodermal dysplasia. Similarly, mutations in a number Genes were selected based on their involvement in human dis- of genes have been implicated thus far in AI, of which 10 cause eases with orodental phenotypes, mutation in animal models ENAM WDR72 KLK4 an exclusively dental phenotype ( , , , presenting orodental disorders,48 49 expression in the develop- AMELX MMP20 FAM83H AMBN ITGB6 SLC24A4 , , , , , and ing mouse tooth50 and known role in tooth development. Two c4orf26 21–30 LTBP3 ), some cause syndromic disease with AI ( , versions of the gene panel were developed, v1.0 and v2.0. v1.0 FAM20A CNNM4 ROGDI STIM1 FAM20C 31–36 , , , and ), and yet was used for patients V1.01–V1.16, whereas v2.0 was used for COL17A1 others account for isolated and syndromic AI ( , patients V2.01–V2.95. Complementary RNA capture probes LAMA3 LAMB3 DLX3 37–40 , and ). Indeed, the pattern of inherit- were designed against all coding exons and 25 bp of flanking ance and penetrance associated with each gene also varies. This intronic sequence in order to cover splice junctions of these wide range of heterogeneity can render genetic diagnosis genes using the SureDesign portal (https://erray.chem.agilent. challenging. com/suredesign, Agilent, USA). Yet, the early molecular diagnosis of orodental disorders is important as it can improve patient care. For instance, mutations in AXIN2 that cause STHAG have been shown to predispose Library preparation, sequencing and data analysis carriers to colorectal cancer.12 Early diagnosis of AXIN2 muta- Targeted regions were captured using a Custom SureSelectXT2 tions can hence alert clinicians to counsel patients to have in-solution target enrichment kit (Agilent) and libraries were regular colonoscopies. Similarly, the identification of mutations prepared for sequencing (2×100 bp) on the HiSeq2500 ’ in FAM20A in patients presenting with AI can prompt a renal (Illumina, USA) following the manufacturer s instructions. For investigation for the management of nephrocalcinosis.41 v1.0, 16 samples were multiplexed per lane for sequencing, fl Targeted next-generation sequencing (NGS) has proven whereas for v2.0, 32 samples were multiplexed per ow-cell extremely beneficial clinically for the molecular diagnosis of a lane. Read alignment, and variant calling and annotation were fl number of genetically heterogeneous disorders, such as hearing performed using standard methods. Brie y, reads were aligned – loss, mitochondrial disease, intellectual disability (ID), neuro- to the GRCh37 reference genome using Burrow Wheeler – 51 muscular disorders and Bardet–Biedl syndrome.42 46 Better aligner (v0.7.5a) ensuring tagging of multi-mapped reads, and coverage, lower cost and relative ease of data interpretation have duplicates were marked with Picardv1.102 (http://picard. made it more commonplace for routine clinical use than whole- sourceforge.net). Indel realignment, base quality score recalibra- tion and variant calling were performed with the GATK Toolkit exome sequencing (WES) and whole-genome sequencing 52–54 47 v3.1 using hard-filtering parameters. Variants were anno- (WGS). 55 We have developed the first targeted NGS panel for the tated using snpEffv.3.4. Variant frequencies were compared with an internal exome database and prioritised using molecular diagnosis of isolated and syndromic orodental disor- 56 ders. We demonstrate the utility of this panel in the molecular VARank. Variants were prioritised by allele frequency (<1% in the Single Nucleotide Polymorphism database (dbSNP137), diagnosis of a variety of orodental disorders. In a cohort of 103 57 patients (101 unrelated) without a known molecular diagnosis 1000 Genomes database, Exome Variant Server (EVS) data- 58 http://jmg.bmj.com/ referred to the Reference Centre for Rare Diseases with base and our internal database, except for non-syndromic