Tooth development is a process consisting of formation and eruption steps, that involves a complex interplay of the epithelium with the neural crest derived mesenchyme. Most of the genes involved in tooth development include signalling molecules belonging to BMP, FGF, SHH and WNT pathways. While the role of some genes in tooth development has been elucidated, several aspects related to molecular bases of the odontogenesis are still under investigation. Emerging evidence suggests that also microRNAs (miRNAs) may exert important functions in human dental tissues, whose perturbation could underlie both dental agenesis and eruption process. Dental agenesis is one of the most common dental abnormalities, and dental eruption disturbances are more frequent than the agenesis, with impaction of the maxillary permanent canine as the second most frequent tooth impaction after third molar.
The aim of this project is to study the role of miRNAs in dental anomalies, specifically in canine eruption process. We will focus on DNA variations in miRNAs as genetic determinants of eruption anomalies, both in familiar and sporadic cases, through a combined approach of in silico and experimental studies. The analysis will be performed on already available cases, i.e. a family segregating a phenotype involving the maxillary canines characterized by incomplete penetrance and variable expressivity, and 50 sporadic cases, and will be extended to cases recruited during the project.
This approach will allow to identify and select candidate variants in miRNAs that potentially affect their activity and expression levels, through the alteration of biogenesis and/or targeting.
The identification of functional variants in miRNAs will allow to elucidate molecular bases of tooth agenesis which is a fundamental requirement for genetic counselling and also for the clinical management of dental anomalies, in order to prevent occlusion problems, particularly during developing dentition in children.
The aetiology of canine agenesis and impaction is still largely unknown, with supposed genetic and environmental factors, exerting a role in the pathogenesis. Recently, multiple lines of evidence highlighted the role of few specific cell signalling pathways as crucial mediators of tooth development and tooth agenesis and inclusion (Yin 2015). This evidence has been recently confirmed by the applicant through the clinical and genetic study of an Italian family with several members with maxillary canine anomalies, including canine agenesis, either monolateral or bilateral, canine impaction and canine ectopic eruption, characterized by incomplete penetrance and variable expressivity (Barbato 2018).
The Next Generation Sequencing (NGS) approaches gave a great impulse to the research of the molecular bases underlying tooth anomalies and, recently, also to the canine anomalies, including agenesis and eruption. Specifically, targeted and Whole Exome Sequencing (WES) approaches confirmed the role of cell signalling pathways, as EDA, WNT and NF-KB and expanded the number of genes involved in tooth development and movement, whose mutations cause canine anomalies spectrum. To date, only very few papers reported the use of NGS strategy to study genetic aetiology of orodental anomalies, in particular canine anomalies (Prasad 2016; Ockeloen 2016; Massink 2015; Salvi 2016; Yamaguchi 2017; Barbato 2018).
Additionally, these evidences suggest that other genetic, epigenetic and environmental contributory factors may also be involved and that integrative and multidisciplinary approaches are warranted to shed light on the pathogenesis of tooth anomalies.
In this context, microRNAs (miRNAs) have recently emerged as crucial post-transcriptional regulators of tooth development and eruptions processes (Sehic 2017). Variants in miRNA genes can have large effects, due to their role as a fine-tuner of gene expression and translation of their downstream targets. In line with this, different miRNA variants have been found to be involved in tooth anomalies. Though some aspects of miRNA biogenesis and functioning have already been unraveled, more investigations are needed to dissect the regulation of miRNAs in processes as the epithelium and neural crest derived mesenchyme signalling, alveolar bone resorption and dental follicle formation.
Dissecting the genetic component underlying canine anomalies is a challenging task, as there are several limitations that influence causative factors identification, for example complex inheritance, reduced penetrance, variable expressivity, and the occurrence of sporadic cases that makes linkage analysis unfeasible.
The applicant has recently developed a workflow of WES data analysis, that is able to retrieve information on the miRNA fraction of the human genome from WES data, allowing to re-analyze all the WES already performed in house including exomes of the family with several members affected by canine anomalies spectrum (Barbato 2018).
To date, miRNA genetic variability and the functional effect of single nucleotide variants (SNVs) is still poorly characterized, due to the paucity of dedicated tools and the scattered annotation resources that make the interpretation of miRNAs sequence variants still a challenging task. To address this issue, the applicant developed a deleteriousness scoring system, to specifically predict the functional effect of variants in miRNAs, potentially affecting biogenesis and targeting. Moreover, to annotate miRNA variants in terms of biological information the applicant developed a database with all the possible 458925 allelic variants at each single nucleotide positions of 1869 miRNAs hairpin precursor transcripts annotated in miRBase (Kozomara 2019), based on conservation, miRNAs function, expression, animal models and phenotype. These tools will allow to identify variants in miRNAs both in familiar and sporadic cases and to select candidate variants that potentially affect miRNAs' activity and expression levels.
A crucial aspect that influences feasibility of this project is represented by the patients' cohort recruitment. The Department of Orthodontics at Sapienza University of Rome directed by Prof. Ersilia Barbato, is a reference center for patients with canine anomalies. The prevalence of canine impaction at the Orthodontics Unit is 9%, with 75% of canine impaction anomalies affecting maxillary canines (Mercuri 2013). This prevalence gives the opportunity to investigate the genetic component of canine anomalies on a large and well characterized samples and to selected properly matched control subjects.
The identification of functional variants in miRNAs will allow to elucidate molecular bases of tooth agenesis which is a fundamental requirement for genetic counselling and also for the clinical management of dental anomalies, in order to prevent occlusion problems, particularly during developing dentition in children.