Autosomal-dominant Ankyloglossia and Tooth ...

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Campus Universitário Darcy Ribeiro, Faculdade de Ciências da Saúde .... families with Kindlers or van der Woude syndrome (Hacham-. Zadeh et al., 1985; ...
RESEARCH REPORTS Clinical

A.C. Acevedo1*, J.A.C. da Fonseca1, J. Grinham2, K. Doudney2, R.R. Gomes1, L.M. de Paula1, and P. Stanier2 1

Oral Care Center for Inherited Diseases, University Hospital of Brasilia, Department of Dentistry, School of Health Science, University of Brasilia, Brazil; and 2Institute of Child Health, University College London, 30, Guilford Street, London,WC1N 1EH, UK; *corresponding author, Campus Universitário Darcy Ribeiro, Faculdade de Ciências da Saúde, Departamento de Odontologia, Asa Norte, 7091090, Brasília, DF, Brazil, [email protected]

Autosomal-dominant Ankyloglossia and Tooth Number Anomalies

J Dent Res 89(2):128-132, 2010

ABSTRACT

INTRODUCTION

Ankyloglossia is a congenital oral anomaly characterized by the presence of a hypertrophic lingual frenulum. It frequently accompanies X-linked cleft palate and is sometimes seen alone due to mutations in the gene encoding the transcription factor TBX22, while knockout of Lgr5 in the mouse results in ankyloglossia. The aim of the present study was to characterize the phenotype and to verify sequence variations in the LGR5 gene in a Brazilian family with ankyloglossia associated with tooth number anomalies. Twelve individuals of three generations were submitted to physical, oral, and radiographic examinations and molecular analysis. Eight had ankyloglossia with various degrees of severity. Six also had hypodontia in the lower incisor region; one had a supernumerary tooth in this region, and another had a supernumerary tooth in the lower premolar region. The characterization of this family determined an autosomal-dominant inheritance and excluded the LGR5 gene mutations as being involved in the pathogenesis of this condition.

A

KEY WORDS: ankyloglossia, tooth agenesis, supernumerary teeth, LGR5 gene.

DOI: 10.1177/0022034509356401 Received August 5, 2008; Last revision July 21, 2009; Accepted September 22, 2009

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nkyloglossia (OMIM 106280), from the Greek words “agkilos” for crooked and “glossa” for tongue, commonly known as ‘tongue-tie’, is a congenital oral anomaly characterized by the persistence of the lingual frenulum as an anatomical abnormality. The lingual frenulum recedes as a natural process during the child’s growth and development, and occurs between 6 mos and 6 yrs of age. Ankyloglossia severity can range from a slight abnormality with no clinical significance to a tongue completely fixed to the floor of the mouth. It may also be an isolated finding, or can be found as a part of certain malformation syndromes (Mintz et al., 2005). The prevalence is reported at around 3-4% of babies and shows a male preponderance, with ratios varying from 1.5:1 to 2.6:1 (Wallace, 1963; Ballard et al., 2002; Hall and Refrew, 2005; Ricke et al., 2005). The shortened frenulum may restrict movement of the tongue, affecting breastfeeding and speech during infancy, and can influence social behavior and self-confidence of children and adolescents (Messner and Lalakea, 2000). Several methods have been proposed to classify the severity of ankyloglossia according to either anatomical or functional criteria (Fletcher and Meldrum, 1968; Ruffoli et al., 2005). However, at present, a unified classification system is still lacking. Meanwhile, management of ankyloglossia in newborns or young infants is also the subject of ongoing controversy (Wright, 1995; Ballard et al., 2002; Messner and Lalakea, 2002). Little is known about the underlying pathogenesis of ankyloglossia. In the literature, 2 genes have been associated with the condition. The first encodes the transcription factor TBX22, which when mutated results in X-linked cleft palate with ankyloglossia (CPX; OMIM303400) (Braybrook et al., 2001, 2002; Marçano et al., 2004). More recently, knockout mice with a targeted deletion of a G-protein-coupled receptor 5 (Lgr5) gene exhibited a phenotype of ankyloglossia (Morita et al., 2004). Lgr5 encodes a 907aa protein, which is located on mouse chromosome 10qD2. All Lgr5 null mice died within 24 hrs due to suckling defects. Histological analyses revealed fusion of the tongue to the floor of the oral cavity in the mutant newborns, and immunostaining of Lgr5 expression in the epithelium of the tongue and in the mandible of the wild-type embryos, suggesting a role of the Lgr5 signaling pathway in proper tongue development (Morita et al., 2004). We reasoned that the human orthologous Lgr5 gene was a candidate for possible involvement with the phenotype in this family. LGR5 maps to human chromosome 12q21.1, spanning 144,809 bp of the genome, and is organized into 18 coding exons. The aim of the present study was to describe the phenotype of a Brazilian family with autosomal-dominant ankyloglossia associated with tooth

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Table 1. Oligonucleotide Primers Used for Amplification and Sequencing of the LGR5 Gene Primer Name EX1F EX1R EX3F EX3R EX5F EX5R EX7F EX7R EX9F EX11R EX13F EX13R EX15F EX15R EX17F EX17R EX18F2

Sequence (5′ > 3′) CTGAGTTGCAGAAGCCCAC CTTGGACGAACCTTCCTTC ATGACTTCCACATGAAGAGG AGTGTGATATGGCTGTATGG AAATGCTCCAGATGTCAGGG CCCATTTGAGCTTTGAAGAG GGGTTCCTTGGAATAGCATG TACTGGTATTTGCTAGAGATAG CCTGAAACCAAGGAATTGGG TCATGTCTGCCATTTCACCC TGATTAAGGGCAGGTAGTCC GTCCCACCACAATAGTACAC AGAGTAAGGACCCTGCTTAG CAAAAGTGTCTCACGACCTC ACTCTCCACTCTGGGATTTG TGGACTCAGAATCAGTGGTG CTACTGCAATTTGGACAAGGG

number anomalies. In addition, LGR5 exon sequences and flanking regions were investigated for a possible causative mutation in this family.

MATERIALS & METHODS Participants and Clinical Examination The Ethics Board of the Brazilian Ministry of Health-CONEP approved this study, and written informed consent was obtained from all participants. A questionnaire was administered, and complete physical and oral examinations were performed on all family members who agreed to participate in the study. Panoramic radiographs and photographs were also performed. Tooth agenesis was considered when a tooth was not visualized clinically or in the radiographs with no report of previous extraction. Supernumerary teeth were diagnosed when an additional tooth was visualized clinically and/or radiographically.

Genetic Analysis Venous blood samples were collected from affected individuals of the family, and genomic DNA was isolated from whole blood by means of the Wizard Genomic DNA purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. PCR primers designed for intronic sequences flanking the 18 exons of the LGR5 gene (Table 1) were used to amplify DNA templates from III2, III3 (affected brothers of the proband), III1 (unaffected wife of III2), and two other unrelated individuals for sequence analysis. An annealing temperature of 57°C was used for each primer pair except for EX9F and EX11R (56°C), which were used to amplify and sequence exons 9, 10, and 11 in a single fragment. Sequencing was performed with BigDye Terminators on an ABI3100 genetic analyzer (Applied Biosystems, Warrington, UK) Sequence traces were analyzed with the Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA).

Primer Name

Sequence (5′ > 3′)

EX2F EX2R EX4F EX4R EX6F EX6R EX8F EX8R EX12F EX12R EX14F EX14R EX16F EX16R EX18F1 EX18R1 EX18R2

CAATTCATTTGTCAAGCAGGG AAATTGTTGGTGCTGACAGG TCACAGGAATTTCAGCTCCC GGTATGACTCCCAACCAAGG GTTAAGTGTGGTCTGTGCAG CAAACCAAACAAAGTCTGGC TTATTGCCACTGTGGTCAGG AGTGTTCAGCTAGAACCTGG TGTTCAGAGTCTGCCTTTGG CAGTGACCCTGAGAAATGTG TCTGAAGGGCAGTTTGATAAG TGCATATCCAGCTTCTTCAAC CCAAAGGTAGAATAATTGGGTG CCATGTGTTCCACTGAACAC GACCATTATCTCTTGGCATCC GTTTTACCATAGAGCAGTCCC TCACTCTCAAGTATCAAGAGG

RESULTS The proband (III:4), a 21-year-old female, was referred to the Oral Care Center for Inherited Diseases, University Hospital of the University of Brasília, Brazil, presenting hypertrophy of the lingual frenulum and labial frenulum. The individual’s medical history revealed no evidence of systemic disease except for asthma. Family history showed that the proband was the younger daughter of nonconsanguineous parents (Fig. 1). The intra-oral examination revealed a severely hypertrophic lingual frenulum in the anterior region of the dental arch (Fig. 2), hypertrophic lower labial frenulum, and the absence of 3 permanent teeth, the lower central incisors and the lower left lateral incisor. The radiographic examination confirmed agenesis of the lower anterior teeth corresponding to the region of the frenulum malformation diagnosed as ankyloglossia (Fig. 2). Apart from ankyloglossia, a hypertrophic lower labial frenulum, and tooth agenesis, no other soft-tissue alterations or dental anomalies were observed. In total, 12 family members were examined, and the oral findings are summarized in Table 2. The examinations revealed that eight individuals (II:2; II:10; II:12; III:1; III:2; III:3; III:8, and IV:1) presented ankyloglossia and hypertrophy of the labial frenulum with various degrees of severity. These individuals did not report speech difficulty or breastfeeding problems. The proband also reported that two additional family members had similar features (II:4 and III:6), but refused to participate in the study. Five individuals (II:2; II:10; II:12; III:3, and IV:1) had been subjected to previous partial or total frenectomies at the time of the examination. Individuals III:3 and IV:1 presented bifid tongue resulting from unsuccessful surgery. The proband’s mother’s (II:2) examination showed ischemia of the central region of the tongue when protruded. The proband’s brother (III:1) presented labial commissure pits (Fig. 2). Several individuals with ankyloglossia also had tooth number anomalies. The distribution of tooth number anomalies among family members is shown in Fig. 1. Five of the

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and/or shortened frenulum, leading to the ankyloglossia phenotype (Morita et al., 2004; Yamane, 2005). In this study, a Brazilian family with ankyloglossia and tooth anomalies was characterized. All affected individuals presented with severe hypertrophic lingual frenulum and tooth anomalies. In this family, inheritance appears to be autosomal-dominant. Few other reports are available that define the mode of inheritance of this condition. This is probably because ankyloglossia varies markedly in severity, and without universal uptake of a classification system, the resulting data are highly heterogeneous. As a consequence, studies reporting systematic family data across a number of families with and without the condition are not available. Several descriptions Figure 1. Pedigree and family members permanent tooth number anomalies. (A) Family pedigree. (B) Tooth of familial ankyloglossia have number anomalies in the examined family members. Tooth agenesis is represented by black teeth and supernumerary by gray teeth. been published, including a Dutch family in which 13 individuals over three generations had ankyloglossia, with male-to-male transmission reported (Keizer, 1952). affected individuals had tooth agenesis in the region of the More recently, a study of a Finnish family with isolated ankyloglosfrenulum anomaly (II:2; III:1; III:3; III:8, and IV:1). Besides sia, inherited as an autosomal-dominant trait with incomplete pentooth agenesis, two of the individuals had supernumerary etrance, also suggested a male predominance (Klockars, 2007). teeth. A deciduous mesiodens in the region of the frenulum However, these reports are not sufficiently extensive to allow conanomaly was observed in individual IV:1 (Fig. 2), and clusions to be drawn about male-to-female ratios. another supernumerary tooth was identified radiographically Ankyloglossia has also been noted as an occasional feature in between the second lower premolar and first lower molar left families with Kindlers or van der Woude syndrome (Hachamof individual III:8. Zadeh et al., 1985; Burdick et al., 1987); however, the best-studied Sequence analysis of affected individuals was entirely concondition including ankyloglossia is X-linked cleft palate and ankysistent with that of unrelated individuals and the published loglossia (CPX). In families with CPX, the tongue defect was noted genome sequence (http://genome.ucsc.edu/). Several coding as a common isolated feature in carrier females, but was present SNPs have been reported for LGR5, all of which are fairly infrealong with the cleft palate in affected males (Moore et al., 1987; quent (http://www.ncbi.nlm.nih.gov/sites/entrez). None was Bjornsson et al., 1989; Gorski et al., 1992). Missense, splice site, present in this family, although several unrelated control indiand nonsense mutations have been identified in TBX22 in families viduals were found to be heterozygous for a missense change with CPX (Braybrook et al., 2001). Expression of this T-box tran(rs17109924) in exon 18. This analysis excluded coding region scription factor was shown both in the developing palatal shelves changes, but did not rule out promoter region or deep intronic and in the base of the tongue, consistent with the CPX phenotype variants that might influence LGR5 expression or splicing. (Braybrook et al., 2002). Confirmation that TBX22 mutations are a significant cause of both cleft palate and ankyloglossia, either as DISCUSSION isolated or combined defects, has come from several recent studies The sublingual frenulum is a fold of mucosa connecting the midline (Marçano et al., 2004; Andreou et al., 2007; Suphapeetiporn et al., of the ventral side of the tongue to the floor of the mouth. Studies 2007). In these studies, males frequently exhibited cleft palate and in animal models have shown that, during embryonic development, ankyloglossia together (78%), as did a smaller percentage of carrier the tongue is formed from the foregut endoderm, and by E13 the females; however, a range of severity was observed, including distal end of the tongue is freed from the floor of the mouth. complete cleft of the secondary palate, submucous cleft, bifid Programmed cell death and resorption of the developing skeletal uvula, absent tonsils, or high vaulted palate (Marçano et al., 2004). muscle in the ventral anterior region free the tongue, and, normally, Ankyloglossia as the sole phenotype is rare in males (4%), but a thin tissue band, the lingual frenulum, remains as the only attachfrequent in female carriers (45%). Mutations within single families ment. Disturbances of this process result in an anteriorly extended could result in either cleft palate only, ankyloglossia only, or both,

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indicating that these defects are distinct parts of the phenotypic spectrum (Marçano et al., 2004; Stanier and Moore, 2004; Pauws and Stanier, 2007). A possible causative role for TBX22, in this Brazilian family, was excluded by two instances of male-to-male transmission, which ruled out X-linked inheritance and suggested autosomal-dominant inheritance. However, it is possible that a gene downstream of this transcription factor is involved. LGR5 represents one of the only other candidate genes for ankyloglossia described to date. In the mouse, the Lgr5 homozygous knockout has ankyloglossia as the primary phenotype, although within the first 24 hrs after birth, the gastrointestinal tract becomes distended with the ingestion of air during the animal’s unsuccessful attempts to suckle (Morita et al., 2004). This combination is lethal in the mouse, which is in stark contrast to the effects of ankyloglossia in humans. The sequence analysis presented in this study failed to find any evidence pointing to a role for LGR5 in the phenotype found in this family. The majority of the affected individuals also had tooth agenesis, and one individual had a supernumerary tooth in the malformation region, suggesting that the tooth anomalies might be a consequence of frenulum anomaly. However, we cannot exclude a genetic basis. One individual also had supernumerary permanent teeth outside the ankyloglossia region, which might suggest that the disruption of the molecular regulation of the lingual frenulum development, in this family, might also have affected tooth development. To our knowledge, there are no previous reports concerning familial ankyloglossia associated with tooth number anomalies. Previous analyses of the cumulative data from mouse and human genetic studies have shown that several transcription factors are involved in the pathogenesis of syndromic and nonsyndromic tooth agenesis (Matalova et al., 2008). Whereas very little is known about supranumerary teeth (D’Souza and Klein, 2007), mutations in MSX1 and PAX9 genes have been identified in families with autosomal-dominant hypodontia and oligodontia (Matalova et al., 2008). In addition, a missense mutation in a Finnish individual with oligodontia and colorectal cancer has been identified (Lammi et al., 2004). However, these mutations represent a small percentage of affected individuals. Genes associated with craniofacial syndromes such as IRF6, FGFR1, and TGFB3 are putative candidates, since it has been shown that they are also associated with non-syndromic tooth agenesis. Other candidate genes expressed in developing teeth germs and knockout models showing a tooth agenesis phenotype include Lef1, Dlx1, Dlx2, Barx, and Lhhx6 (Thesleff, 2003). The presence of supernumerary teeth is a relatively rare condition characterized by the presence of more than the normal complement of 20 deciduous and 32 permanent teeth. Supranumerary teeth are often observed in syndromes such as cleidocranial dysplasia (CCD) syndromic cleft lip and palate, Gardner syndrome, and Nance Horan syndrome. Mutations in the RUNX2 gene have been shown to cause CCD. Chromosomal translocations, deletions, insertions, nonsense, and splice-site mutations, as well as missense mutations of the RUNX2 gene, have been described in persons with CCD (Otto et al., 2002). At present, no reports concerning the genetic and molecular

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Figure 2. Clinical and radiographic findings in the examined family members. (A) Proband (III:4), with ankyloglossia and absence of lower central incisors and lower left lateral incisor. (B) Bifid tongue and supernumerary tooth in the primary dentition (IV:1). (C) Hypertrophy of labial frenulum (III:1). (D) Labial commissure pits (III:1). (E) Panoramic radiographic of proband (III:4), showing absence of lower central incisors and lower left lateral incisor (arrow). (F) Panoramic radiographic showing the absence of lower incisors in the permanent dentition and supernumerary tooth in the primary dentition (IV:1).

defects that lead to non-syndromic supernumerary tooth development are available. In this study, LGR5 gene mutations in the coding region were excluded as a causative role in a family with autosomal-dominant ankyloglossia and tooth number defects. However, it does not rule out the possibility that mutations in the promoter region or in the deep introns might be responsible for this syndrome. Nevertheless, LGR5 remains an interesting candidate to investigate in future studies involving cohorts of persons with ankyloglossia. Considering the presence of tooth anomalies in this family, an analysis of gene candidates associated with the pathogenesis of tooth agenesis and supranumerary teeth should also be undertaken.

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Table 2. Summarized Oral Findings of Family Members Examined Pedigree Number

Gender

Age (yrs)

Ankyloglossia

Hypertrophic Labial Frenulum

II:2 II:5 II:10 II:12 III:1 III:2 III:3 III:4

F F M M M F M F

49 34 40 39 29 25 23 21

+ + + + + +

* * * + * +

III:8 III:14 III:15 IV:1

F F M M

17 15 11 7

+ +

+ *

*

Missing Tooth Lower incisors

-

Lower incisors Upper left third molar Lower central incisors Lower central incisors, lower left lateral incisor Lower incisors Lower incisors

Supernumerary Teeth + +

Individual submitted to partial or total frenectomy at the time of the examination.

ACKNOWLEDGMENTS The work was supported by FINATEC, UnB. We are grateful to those who kindly agreed to participate in this study.

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