Rethinking the Genetic Etiology of Nonsyndromic Tooth Agenesis

Abstract

Purpose of Review:

Genetic studies in humans and animal models have improved our understanding of the role of numerous genes in the etiology of nonsyndromic tooth agenesis (TA). The purpose of this review is to discuss recently identified genes and loci potentially contributing to TA.

Recent Findings:

Despite research progress, understanding the genetic factors underlying nonsyndromic TA has been challenging given the genetic heterogeneity, variable expressivity, and incomplete penetrance of pathogenic variants often observed associated with the condition. Next generation sequencing technologies have provided a platform for novel gene and variant discoveries and informed paradigm-shifting concepts in the etiology of TA.

Summary:

This review summarizes the current knowledge on genes and pathways related to nonsyndromic TA with a focus on recently identified genes/variants. Evidence suggesting possible multi-locus inheritance for TA is also presented.

Introduction

Tooth development is a genetically-regulated process characterized by a series of sequential and reciprocal epithelial-mesenchymal interactions that drive tooth morphogenesis as well as differentiation of tooth-specific cell types.[1] Four major signaling pathways (BMP, FGF, SHH, WNT) and various transcription factors are key orchestrators of tooth development (Figure 1). Disturbances at any stage of tooth development may lead to a plethora of dental anomalies, of which tooth agenesis (TA) is the most widely recognized. [2]

Fig. 1.

Schematic representation of tooth development stages. Sequential and reciprocal interactions between epithelium and mesenchyme are tightly regulated by various signaling pathways (orange) and transcription factors (green). Disturbances in the expression of molecular players the initiation stage of tooth development results in tooth agenesis.

TA is defined as the developmental failure of a tooth due to perturbations in the initiation stage of tooth development.[2] Agenesis of primary teeth is rare, however, there is a strong correlation between agenesis in the primary dentition and permanent dentition.[3] TA affects approximately 200 million individuals worldwide and can occur as a feature of many craniofacial syndromes, although it is more frequently found as a nonsyndromic, isolated trait segregating in families or as a new event. Autosomal dominant, autosomal recessive, or X-linked patterns of inheritance have been reported.[4, 5] Depending on the number of missing teeth, TA can be referred to as hypodontia (1 to 5 teeth missing), oligodontia (≥6 teeth missing) (Figure 2), or anodontia (all teeth missing), the latter being primarily associated with syndromic forms.[6]

Fig. 2.

Panoramic radiograph of 8-year old child presenting with severe tooth agenesis (oligodontia). Asterisks denote location of permanent teeth missing (asterisks).

The prevalence of TA, excluding third molars, has been reported to range between 1.6% to ~10%, depending on severity (hypodontia or oligodontia) and population studied. When third molars are included in the missing teeth count, the prevalence of TA reaches 25%.[4] Hypodontia is more common, whereas oligodontia is rare occurring in <1% of the population. The most commonly affected teeth are mandibular second premolars followed by the maxillary lateral incisors, and maxillary second premolars. It is also generally accepted that the prevalence of TA varies by sex, with females being more affected than males in a 3:2 ratio.[4]

TA imposes significant functional, esthetic, and financial burdens on affected individuals and families. Clinical management of TA is challenging and requires a multidisciplinary team to perform multiple orthodontic, rehabilitation, and/or surgical interventions to close the missing spaces and restore masticatory function and esthetics. TA is often accompanied by delays in permanent tooth development and additional dental anomalies including microdontia, taurodontism, transposition, and ectopic positioning of erupted teeth. The impact on oral health-related quality of life can be significant and depend on the affected individual’s age, severity of TA, and the presence of additional dental anomalies. Treatment strategies to mitigate the functional and cosmetic consequences of TA vary and often include space management and maintenance, alignment of existing teeth, occlusal bite corrections, among other oral rehabilitation procedures that often come with a high cost.[4]

Compelling evidence supports a predominantly genetic etiology for TA.[5] Studies in animal models have elucidated the role of numerous genes during tooth development thereby facilitating molecular diagnosis of TA in affected individuals.[2, 3] Despite research progress, understanding the genetic factors underlying nonsyndromic TA has been challenging. Genetic heterogeneity, variable expressivity, pleiotropic effects of genes, and incomplete penetrance may act as confounders complicating causal gene identification. Further, it has been proposed that the missing heritability of TA relies on mutations in genes yet unknown to tooth development, and/or on combined effects of multiple pathogenic variants.[3, 7]

In this review, we summarize the current knowledge on genes related to nonsyndromic TA and highlight the recent contributions of next-generation sequencing studies in advancing knowledge about the etiology of the condition.

Genes involved in nonsyndromic TA

Defects in genes involved in the process of odontogenesis have been reported as etiologic for nonsyndromic TA, often following clues from animal models or syndromic presentations (e.g., oral-facial cleft and ectodermal dysplasia syndromes).[5, 8] While the scope of this review is limited to the genetic etiology of nonsyndromic TA, genes involved in syndromic TA and associated phenotypes have been extensively discussed.[2, 3, 5] Genes in which variations have been reported in nonsyndromic TA individuals are presented in Table 1.

Table 1.

Genes in which mutations have been reported in nonsyndromic TA individuals.

Gene	Location	Mode of Inheritance	Phenotype(s)		Missing teeth frequently associated	Gene discovery approach**
ANTRX1	2p13.3	AR	Oligodontia	----	mandibular incisors and canines	WES
ARHGAP15	2q22.2-q22.3	Unknown	----	Hypodontia	Not reported	GWAS
ASCL5	1q32.1	Unknown	----	Hypodontia	Not reported	GWAS
AXIN2	17q24.1	AD	Oligodontia	Hypodontia	maxillary and mandibular lateral incisors, premolars, and first molars	Positional cloning, sanger sequencing
ATF1	12q13.13	Unknown	Oligodontia	Hypodontia	maxillary and mandibular lateral incisors, premolars, and molars	SNP genotyping
BCOR	Xp11.4	Complex	Oligodontia	----	maxillary canines, second premolar and second molar, mandibular central and lateral incisor, second premolar	WES
CACNA1S	1q32.1	Unknown	----	Hypodontia	Not reported	GWAS
CASC8	8q24	Unknown	Oligodontia	Hypodontia	maxillary and mandibular lateral incisors, premolars, and molars	SNP genotyping
COL17A1	10q25.1	Complex	----	Hypodontia	maxillary lateral incisors, mandibular central and lateral incisors	WES
DKK1	10q21.1	Complex	----	Hypodontia	maxillary lateral incisors, mandibular central and lateral incisors	WES
DUSP10	1q41	Unknown	Oligodontia	Hypodontia	maxillary and mandibular lateral incisors, premolars, and molars	SNP genotyping
EDA	Xq13.1	XL	Oligodontia	Hypodontia	maxillary and mandibular central and lateral incisors	sanger sequencing
EDAR	2q13	AD	Oligodontia	Hypodontia	mandibular second premolars, maxillary lateral incisors	sanger sequencing
EDARADD	1q42.3-q43	AD	Oligodontia	Hypodontia	canines	sanger sequencing
FAM49A	2p24.2	Unknown	----	Hypodontia	maxillary lateral incisors	GWAS
FGF3	11q13.3	Unknown	----	Hypodontia	maxillary lateral incisors	SNP genotyping
FGF10	5p12	Unknown	----	Hypodontia	maxillary lateral incisors, premolars	SNP genotyping
FGFR1	8p11.23	AD	----	Hypodontia	maxillary and mandibular incisors and premolars	SNP genotyping
FGFR2	10q26.13	Unknown	----	Hypodontia	premolars	SNP genotyping
FOXI3	2p11.2	Unknown	----	Hypodontia	Not reported	GWAS
FOXP1	3p13	Unknown	----	Hypodontia	maxillary lateral incisors	GWAS
GLI2	2q14.2	Unknown	----	Hypodontia	mandibular incisors	SNP genotyping
GREM2	1q43	AD	Oligodontia	Hypodontia	maxillary and mandibular lateral incisors, premolars and molars	sanger sequencing
IRF6	1q32.2	AD	----	Hypodontia	maxillary and mandibular incisors, premolars and molars	SNP genotyping
LAMA3	18q11.2	Complex	Oligodontia	Hypodontia	maxillary lateral incisors and premolars, second molar, mandibular premolars	WES
LAMB3	1q32.2	AR	Oligodontia	----	maxillary lateral incisors and premolars, mandibular canines and first premolar	WES
LHX6	9q33.2	Unknown	----	Hypodontia	premolars	SNP genotyping
LRP6	12p13.2	AD	Oligodontia	Hypodontia	incisors and premolars	WES
MSX1	4p16.3-p16.1	AD	Oligodontia	Hypodontia	mandibular and maxillary premolars and molars, maxillary lateral incisors	sanger sequencing
NOL11	17q24.2	Unknown	----	Hypodontia	maxillary second premolars	GWAS
PAX9	14q12-q13	AD	Oligodontia	Hypodontia	mandibular and maxillary second molars and second premolars	sanger sequencing
SMOC2	6q27	AR	Oligodontia	----	mandibular and maxillary canines, second premolars and molars, and lateral incisors	sanger sequencing
TSPEAR	21q22.3	AD, AR	Oligodontia	----	mandibular and maxillary lateral incisors, canines, second premolars and second molars; maxillary first premolars, mandibular central incisors	WES
WNT10A	2q35	AD, AR, complex	Oligodontia	Hypodontia	mandibular and maxillary lateral incisors, canines, premolars and molars; mandibular central incisors, premolars and molars	sanger sequencing
WNT10B	12q13.12	AD	Oligodontia	Hypodontia	mandibular and maxillary incisors, canines, premolars and molars; mandibular incisors, premolars and molars	sanger sequencing
ZFHX4	8q21.13	Unknown	----	Hypodontia	Not reported	GWAS

^*reflects first gene discovery study.

WES, whole exome sequencing. SNP, single nucleotide polymorphism

AD, autosomal dominant. AR, autosomal recessive

‘Classic’ TA genes

Some of the most widely known genes involved in nonsyndromic TA include the transcription factors MSX1 (Msh homeobox 1) and PAX9 (paired box 9).[2, 3, 9, 10] Autosomal dominant mutations in these genes comprise the first reports of genetic variations in nonsyndromic TA.[11] A missense mutation in MSX1 was found in a multigeneration family affected by TA in which affected individuals lacked second premolars, third molars, and upper lateral incisors. [11] PAX9 was later identified as the second gene involved in TA, where a frameshift mutation was reported as etiologic in a family characterized by the absence of permanent molars.[12] Over the years, more than 50 mutations in MSX1 and PAX9 have been identified in nonsyndromic TA, affecting preferentially posterior teeth (premolars and molars), although a few reports of anterior tooth agenesis exist.[3, 10, 13] In mice, expression of both MSX1 and PAX9 in the dental mesenchyme is critical to initiate and regulate signaling during the transition from bud to cap stage of tooth development; further, both genes act synergistically to regulate the expression of BMP4 (bone morphogenetic protein 4), a member of the transforming growth factor-β (TGF-β) family essential for bone, limb, and tooth development.[14] In addition to inducing TGF‐β/BMP signaling, PAX9 is required for activation of Wnt signaling during craniofacial morphogenesis.[15]

EDA (ectodysplasin-A) mutations result in anhidrotic ectodermal dysplasia and hypohidrotic ectodermal dysplasia, both which include oligodontia as a phenotypic feature [16, 17] EDA binds to its receptor EDAR to activate EDA/EDAR/NF-κB signaling pathway, which is required for normal skin, hair, and teeth formation.[18] Although these genes are more commonly associated with syndromic forms of TA, circa 30 EDA and/or EDAR mutations have been reported in individuals with TA and no other defects of ectodermal nature.[16, 19–21] In mice, loss of Eda confirms the ectodermal and dental phenotypes observed in humans.

WNT pathway genes

The Wnt/β‐catenin signaling pathway regulates many aspects of embryonic development and is spatiotemporally activated throughout tooth development stages, thereby implying its critical role during odontogenesis.[22] (Figure 3).

Figure 3. The canonical WNT/β-catenin signaling pathway.

(A) Wnt signaling is activated via Wnt ligands binding to cell surface receptors Frizzled (FZD) and LRP5/6. which then transduce the signals to a gene complex including AXIN, APC and GSK3 to inhibit β-catenin degradation. Subsequently, β-catenin accumulates in the cytoplasm and translocates into the nucleus to interact with TCF and initiate transcription of target genes. (B) In the presence of DKK, a ternary complex formation between Kremen (KRM), DKK and LRP6 results in depletion of LRP5/6 from the membrane, blocking of Wnt signaling and triggering β-catenin degradation by the gene destruction complex and inhibition of gene transcription (From N. Dinckan, R. Du, L.E. Petty, Z. Coban-Akdemir, S.N. Jhangiani, I. Paine, E.H. Baugh, A.P. Erdem, H. Kayserili, H. Doddapaneni, J. Hu, D.M. Muzny, E. Boerwinkle, R.A. Gibbs, J.R. Lupski, Z.O. Uyguner, J.E. Below, and A. Letra. Whole-Exome Sequencing Identifies Novel Variants for Tooth Agenesis. Journal of Dental Research 97(1):49–59).

Over the years, a number of WNT pathway genes have been implicated in the etiology of nonsyndromic TA. Mutations in AXIN2 (axis inhibition protein 2), a negative regulator of Wnt signaling, were reported as etiologic for TA in a multigenerational family segregating with TA and colorectal cancer, thereby suggesting that TA could be a potential early marker for colorectal cancer in predisposed individuals. [23] In addition, this study showed for the first time, that Axin2 was expressed in the developing mouse craniofacial and dental tissues, specifically the enamel knot and underlying mesenchyme during tooth formation.[23] Several studies have since investigated the association of genetic variation in AXIN2 with sporadic and familial TA, with or without cancer history, although results are not conclusive. In most studies, individual or family history of cancer in studied TA cohorts was not addressed, therefore caution must be taken to avoid interpretation of such association findings as direct evidence of causation.[24, 25]

LRP6 (LDL receptor related protein 6) is a co-receptor in the Wnt/β‐catenin pathway that can bind to various WNT ligands and inhibitors (e.g., DKK, KREMEN) to modulate Wnt/β‐catenin signaling.[26] To date, circa 16 mutations in LRP6 have been reported in individuals with nonsyndromic TA in different populations.[27–33] Most of the identified variants suggest loss-of-function effects although additional functional characterization experiments are required to confirm their impact on gene/protein function. In one study, a splicing variant (c.3607+3_6del) in LRP6 was identified segregating in autosomal dominant form in a multigenerational family with TA. Decreased mRNA expression levels were noted in peripheral blood samples of all affected individuals, indicating nonsense-mediated decay [28]. Intriguingly, the missense variant c.2570G > A (R857H), identified in a TA family and classified as potentially damaging due to its predicted effect on LRP6-Wnt binding did not result in changes in gene transcription, although maturation and phosphorylation levels of LRP6 were noted in the presence of mutant alleles suggesting perturbed Wnt signaling.[34] In mice, Lrp6 expression was noted in the tooth follicle and inner enamel epithelium [29], while homozygous deletion of Lrp6 led to severe skeletal abnormalities and early lethality.[35]

Mutations in WNT10A (wingless-type MMTV integration site family, member 10A) were first identified in individuals with the autosomal recessive disorders odontoonychodermal dysplasia (OODD) and Schopf-Schulz-Passarge syndrome (SSPS), and more recently reported to account for over 50% of the TA-associated genetic variation.[36] More than 60 rare, homozygous and heterozygous variants in WNT10A have been identified in association with nonsyndromic TA.[3, 36] Intriguingly, the same mutation has been associated with both autosomal recessive and autosomal dominant forms of nonsyndromic TA as well as with ectodermal dysplasia, and single heterozygous variants in WNT10A have been identified in approximately 2.5% of individuals without TA (e.g., c.682T>A; F228I) [3]. Consistent with several reports, compound heterozygous in WNT10A were associated with a larger number of missing teeth when compared to single heterozygous mutations. Further, the presence of WNT10A mutations was also frequently reported segregating with mutations in other genes.[28, 37, 38] (see Multilocus inheritance section below). Although no preferential patterns of missing teeth were observed in individual studies [3, 28, 39–42], a recent meta-analysis suggested that the most frequently reported missing teeth patterns associated with WNT10A variation reflect third molars (78%), mandibular premolars (71%) and maxillary lateral incisors (60%).[43] Although strong evidence implicates a role for WNT10A in nonsyndromic TA, its exact role in the pathogenesis of the condition remains unclear. Wnt10a mutant mice do not have missing teeth although defects in tooth morphogenesis have been described.[44, 45] In contrast, downregulation of wnt10a resulted in failure of tooth development in zebrafish embryos.[40]

Additional WNT pathway genes in which variants were associated with nonsyndromic TA include WNT10B (wingless-type MMTV integration site family, member 10B) and DKK1 (Dickkopf WNT Signaling Pathway Inhibitor 1). Rare variants and single nucleotide polymorphisms in WNT10B were identified in three studies of nonsyndromic TA [46–48]. Two studies have suggested DKK1 as a candidate gene for nonsyndromic TA, one of which highlighted a splicing variant in DKK1 segregating with additional missense and splicing variants in LAMA3 (Laminin Subunit Alpha 3) and COL17A1 (Collagen Type XVII Alpha 1), respectively, in a family showing autosomal dominant inheritance of oligodontia phenotypes.[28] (see Multilocus inheritance section below).

Genes identified via genome-wide association study

To the best of our knowledge, a single well-powered genome-wide association study (GWAS) of TA has been conducted, which included 1,944 subjects with congenitally missing teeth and 338,554 controls of European ancestry.[58] Both rare and common genetic variants were identified in association with TA, five of which were located in/nearby genes involved in tooth development and/or additional ectodermal structures (EDA, EDAR, FOXIE3, FOXP1, and LEF1), whereas the remaining associated variants were located around loci with no previous evidence of a role in tooth development (ASCL5/CACNA1S, ARHGAP15, NOL11, and FAM49A).[58] (Table 1)

Genes identified in next generation sequencing studies

The use of next generation sequencing (NGS) and unbiased analysis approaches has made a significant and positive impact on our understanding of the genetic etiology of TA. Recent whole exome sequencing (WES) studies of individuals and families with TA have allowed the identification of novel genes (e.g., ANTXR1, BCOR, COL17A1, DKK1, LAMA3, LAMB3, TSPEAR), as well as novel variants in known genes as etiologic for TA.[3]

ANTXR1 (anthrax toxin receptor 1), in which mutations have been shown to cause GAPO syndrome -- characterized by delayed growth, alopecia, failure of tooth eruption, and optic atrophy -- was recently identified as a novel TA gene in an individual showing agenesis of mandibular incisors and canines and no other structural abnormalities.[49] A rare homozygous ANTXR1 variant (c.1312C>T; R438C) was identified by WES in a consanguineous Turkish family in which the affected individual showed agenesis of mandibular incisors and canines but no other structural abnormalities. Expression of Antxr1 was then detected in the developing mouse craniofacies, particularly in the dental epithelium and mesenchyme at initial stages of tooth development, and later in the enamel organ and dental papilla.[49]

Rare missense variants in LAMA3 (c.1097G>A; R366H; and c.2798G>T; G933V), LAMB3 (Laminin subunit beta-3; c.547C>T; R183C), and a splicing variant in COL17A1 (c.3277+3G>C) were reported as likely pathogenic for nonsyndromic TA in two independent studies.[28, 37] Mutations in these genes cause non-Herlitz-type junctional epidermolysis bullosa, an autosomal recessive skin disorder characterized by the appearance of blisters, erosions, dystrophic nails, enamel hypoplasia, and TA.[50–52] Carriers of heterozygous variants in LAMA3, LAMB3, and COL17A1 have been well described with respect to their amelogenesis imperfecta phenotypes including hypoplastic enamel with the presence of grooves and pits. In mice, deletion of Lama3 resulted in abnormal enamel deposition and disorganized enamel epithelium, whereas deletion of Lamb3 results in early lethality precluding analysis of enamel phenotype. (ref)

Mutations in TSPEAR (thrombospondin-type laminin G domain and EAR repeats) were identified to cause a form of ectodermal dysplasia characterized by facial dysmorphisms, scalp hypotrichosis, TA, and microdontia.[55, 56] These variants were predicted to completely abolish the EAR domains of the TSPEAR protein, or were located within conserved sites of those domains suggesting an important role for TSPEAR in normal development [56]. In two independent studies, a complex insertion/deletion variant (c.1726G>T; 1728delC) and a missense variant (c.1877C>T; P626S; and c) in TSPEAR were identified in families with nonsyndromic TA, although incomplete penetrance was observed in one family.[37, 57] Incomplete penetrance is a common finding in TA families and could be the result of a more complex genotype–phenotype relationship due to variation at other locus/loci, or mutational burden impacting expression of the disease trait associated with monoallelic variation.[37]

Taken together, the results of these next generation sequencing studies revealed the involvement of novel genes in tooth development and potentially etiologic for TA. Additional studies are warranted to confirm the exact mechanisms by which variations in these genes may impact gene/protein function leading to TA.

Multi-locus variation in nonsyndromic TA

Historically, the origin of both syndromic and nonsyndromic TA has been attributed as monogenic, in which a single putatively pathogenic mutation in a gene known to be involved in craniofacial and/or tooth development was deemed etiologic. This was likely due to the genetic research approaches (i.e, candidate gene sequencing) frequently utilized and the establishment of a molecular diagnosis once variation in a known gene was identified. While such efforts enabled the genetic diagnosis for many TA individuals and families, variation in known genes account for ~50–55% of the estimated heritability of TA, indicating that additional genes remain to be identified.[59]

As discussed above, the use of next generation sequencing and unbiased analysis methods allowed the identification of novel genes, variants and variant combinations contributing to nonsyndromic TA. [3] Notably, the use of WES provided evidence that multi-locus variation and oligogenic inheritance is possible in TA, and these observations challenge the notion that a diagnostic investigation is complete after a single variant in a known or novel candidate gene has been obtained.[3] Through independent studies, rare variants in WNT10A were found co-segregating with variants in GREM2, LAMA3, BCOR, or LRP6, in individuals with nonsyndromic TA.[28, 37, 38, 60] Moreover, rare variants in DKK1, LAMA3 and COL17A1 were also found segregating together and likely contributing to the variable TA phenotypes in familial TA.[28] In all these families, a single variant could not explain the TA phenotypes observed suggesting that additional mutational burden or segregation of rare variants at more than one locus could modulate phenotypic expression. Multi-locus variation has been reported as the most likely explanation for a number of Mendelian and complex conditions.[61, 62] TA is a clinically heterogeneous condition with nearly 100 associated loci, hence providing an excellent model for the investigation of mutational burden contributing to complex inheritance patterns. This approach provides a paradigm to maximize identification of the missing genetic liability and explanation for unusual inheritance patterns in TA families.

Challenges and Future Research Directions

TA is characterized by variable expressivity and genetic heterogeneity making genotype-phenotype correlations challenging. A few studies have addressed the patterns of missing teeth with their corresponding genetic etiology although the results vary depending on the population, TA phenotypes (hypodontia versus oligodontia), and if sporadic or familial cases are included.[43, 63] Family-based study designs offer a more powerful approach in studies of complex disorders such as TA because they allow the detection of variant segregation in affected and unaffected family members and the ability to reveal parental-origin allelic effects meanwhile controlling for heterogeneity and population stratification.[64]

Until recently, the field had focused predominantly on protein-coding variation affecting genes involved in craniofacial and/or tooth development, whereas the role of non-coding variants in TA has been less explored. The use of WES facilitated the discovery of novel coding and non-coding variants often located in regulatory regions of the genome as likely pathogenic for TA.[28, 37, 49] 58]. Further, ample evidence now exists to support instances of multilocus variation and potential oligogenic inheritance in TA.[3] While there may be private events happening in private families, as technologies for rare variant detection continue to improve and additional TA genes are identified, additional instances of multi-locus variation are likely to be noted. Therefore, excluding known genes and/or focusing on identification of known genes when conducting NGS studies of TA might preclude proper molecular diagnosis.[3]

Importantly, as new TA variants are identified, functional characterization experiments in vitro and in vivo will be required to determine the mechanisms by which they may disrupt gene/protein function and affect tooth development leading to TA. For many genes, however, the effects of variants predicted pathogenic for TA remain poorly understood. One such example is WNT10A, in which most variants were reported as pathogenic for TA due to their predicted effects on protein folding and/or destabilization of the encoded protein, although the impact of these variants was not always consistent with altered gene/protein function(s).[40, 65] Further, WNT10A variants appear to affect gene/protein function in allele-specific patterns, through direct effects on WNT signaling or indirectly through dysregulated expression of additional relevant genes.[65]

Conclusions

TA is a common abnormality of tooth development with significant functional and esthetic consequences requiring extensive and costly oral rehabilitation treatments. While TA etiology may be monogenic in some cases, evidence supporting multi-locus inheritance is increasing given the wider utilization of next generation sequencing in genetic studies of TA, which show that the segregation of variants in more than one gene can impact signaling activities and affect tooth development. These observations further highlight the importance of employing unbiased filtering and prioritization strategies for next generation sequencing data to maximize gene/variant discovery. Importantly, thorough clinical phenotyping to exclude syndromic forms of TA with subtle manifestations are critical for proper interpretation of genetic findings and improved recurrence risk estimates and genetic counseling.