BackgroundOrodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders.MethodsWe designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption.ResultsWe discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases.ConclusionsWe have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease.Trial registration numbersNCT01746121 and NCT02397824.

Background:We used evolutionary analysis of alkaline phosphatase (tissue nonspecific alkaline phosphatase, TNSALP) to predict missense mutations leading to hypophosphatasia. Results: We found 469 sensitive positions and validated 99% of the 204 known mutations. Conclusion: It is a more powerful method than in silico models to validate missense mutations in TNSALP. Significance: Such an approach should be widely used to support genetic diagnostics.

Molecular evolutionary analysis is an efficient method to predict and/or validate amino acid substitutions that could lead to a genetic disease and to highlight residues and motifs that could play an important role in the protein structure and/or function. We have applied such analysis to amelotin (AMTN), a recently identified enamel protein in the rat, mouse, and humans. An in silico search for AMTN provided 42 new mammalian sequences that were added to the 3 published sequences with which we performed the analysis using a dataset representative of all lineages (circa 220 million years of evolution), including 2 enamel-less species, sloth and armadillo. During evolution, of the 209 residues of human AMTN, 17 were unchanged and 34 had conserved their chemical properties. Substituting these important residues could lead to amelogenesis imperfecta (AI). Also, AMTN possesses a well-conserved signal peptide, 2 conserved motifs whose function is certainly important but unknown, and a putative phosphorylation site (SXE). In addition, the sequences of the 2 enamel-less species display mutations revealing that AMTN underwent pseudogenization, which suggests that AMTN is an enamel-specific protein.

BackgroundAmelotin (AMTN) is an ameloblast-secreted protein that belongs to the secretory calcium-binding phosphoprotein (SCPP) family, which originated in early vertebrates. In rodents, AMTN is expressed during the maturation stage of amelogenesis only. This expression pattern strongly differs from the spatiotemporal expression of other ameloblast-secreted SCPPs, such as the enamel matrix proteins (EMPs). Furthermore, AMTN was characterized in rodents only. In this study, we applied various approaches, including in silico screening of databases, PCRs and transcriptome sequencing to characterize AMTN sequences in sauropsids and amphibians, and compared them to available mammalian and coelacanth sequences.ResultsWe showed that (i) AMTN is tooth (enamel) specific and underwent pseudogenization in toothless turtles and birds, and (ii) the AMTN structure changed during tetrapod evolution. To infer AMTN function, we studied spatiotemporal expression of AMTN during amelogenesis in a salamander and a lizard, and compared the results with available expression data from mouse. We found that AMTN is expressed throughout amelogenesis in non-mammalian tetrapods, in contrast to its expression limited to enamel maturation in rodents.ConclusionsTaken together our findings suggest that AMTN was primarily an EMP. Its functions were conserved in amphibians and sauropsids while a change occurred early in the mammalian lineage, modifying its expression pattern during amelogenesis and its gene structure. These changes likely led to a partial loss of AMTN function and could have a link with the emergence of prismatic enamel in mammals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0329-x) contains supplementary material, which is available to authorized users.