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Tricho-Rhino-Phalangeal Syndrome with Supernumerary Teeth
1 Department of Pediatric Dentistry, Faculty of Dentistry, Chiang Mai University, Thailand; Correspondence: + corresponding author, paul.sharpe{at}kcl.ac.uk
Tricho-rhino-phalangeal syndromes (TRPS) are caused by mutation or deletion of TRPS1, a gene encoding a GATA transcription factor. These disorders are characterized by abnormalities of the hair, face, and selected bones. Rare cases of individuals with TRPS displaying supernumerary teeth have been reported, but none of these has been examined molecularly. We used two different approaches to investigate a possible role of TRPS1 during tooth development. We looked at the expression of Tprs1 during mouse tooth development and analyzed the craniofacial defects of Trps1 mutant mice. In parallel, we investigated whether a 17-year-old Thai boy with clinical features of TRPS and 5 supernumerary teeth had mutation in TRPS1. We report here that Trps1 is expressed during mouse tooth development, and that an individual with TRPS with supernumerary teeth has the amino acid substitution A919V in the GATA zinc finger of TRPS1. These results suggest a role for TRPS1 in tooth morphogenesis.
Key Words: Tricho-rhino-phalangeal syndrome • TRPS1 • tooth development • supernumerary teeth
The Tricho-Rhino-Phalangeal Syndromes (TRPS), first described by Giedion in 1966, are a group of human autosomal-dominant developmental disorders characterized by abnormalities of the hair, face, and selected bones. Individuals with TRPS have sparse fine and slow-growing scalp hair, laterally sparse eyebrows, sparse eyelashes, a bulbous tip of the nose, a large and flat filtrum, a thin upper lip, occasionally large and protruding ears, cone-shaped epiphyses of phalangeal bones, and hip malformations (Giedion, 1966; Giedion et al. 1973). The TRPS have been classified into three types according to their clinical phenotypes and cytogenetic abnormalities. TRPS type I, or Giedion syndrome (TRPS I; OMIM #190350), the prototype of this group of disorders, is caused by deletion or heterozygous mutations in the TRPS1 gene on chromosome 8q24.12 (Momeni et al., 2000; Lüdecke et al., 2001). TRPS1 encodes a large nuclear protein with 9 predicted zinc finger (Zfn) domains, including one GATA-type Znf and a carboxy-terminal IKAROS-like double Zfn. Contrary to other GATA transcription factors that activate transcription, TRPS1 behaves as a strong transcriptional repressor both in vitro and in vivo (Malik et al., 2001, 2002). TRPS II, or Langer-Giegion syndrome (OMIM #150230), is a contiguous gene syndrome and is the result of microdeletion of the contiguous TRPS1 and EXT1 genes. In addition to the TRPS I abnormalities, persons affected with TRPS II exhibit multiple exostoses, predominantly at the ends of the long bones, and some persons with very large deletions tend to have mental retardation (Bühler et al., 1980, 1987; Langer et al., 1984; Yamamoto et al., 1989; Lüdecke et al., 1995, 1999). TRPS III, or Sugio-Kajii syndrome (OMIM #190351), is a form of TRPS with severe short stature and severe brachydactyly as a result of short metacarpals, but without any exostoses, and can be considered a severe form of TRPS I, but not a distinct entity (Niikawa and Kamei, 1986; Lüdecke et al., 2001). In addition to TRPS’ characteristic abnormalities, occasional dental-associated phenotypes have been described, such as microdontia (Goodman et al., 1981), delayed tooth eruption (Hussels, 1971), and malocclusion (Hussels, 1971; Pashayan et al., 1974; Howell and Wynne-Davies, 1986). Interestingly, supernumerary teeth have been observed in several persons with TRPS (for review, see Gorlin et al., 2001). For example, the originally reported individual (Giedion, 1966) had 2 supernumerary incisors. However, the presence of mutations in the TRPS1 gene was not investigated in TRPS associated with supernumerary teeth.
To investigate a possible role of TRPS1 during tooth development, we used two different approaches. We used mouse embryos to study Trps1 expression during tooth development and took advantage of Trps1
In situ Hybridization Wild-type mouse embryos were collected from CD-1 mice. Radioactive in situ hybridization with 35S-UTP labeled RNA probes was carried out as previously described (Wilkinson, 1992) on paraffin tissue sections. Trps1 antisense riboprobe was generated as in Miletich et al.(2005) from a mouse cDNA clone containing a full-length Trps1 cDNA. Slides were counterstained with hematoxylin (Fluka, Poole, Dorset, UK). All animal experiments were carried out in accordance with UK Home Office regulations.
Analysis of Trps1
Mutation Analysis
Trps1 is expressed during Mouse Tooth Development Trps1 expression has previously been found in many developing organs of the mouse (Kunath et al., 2002). This study reported early facial expression of Trps1 in the pharyngeal arches, and later expression in the developing muscles of the tongue and surrounding most of the developing cartilage anlagen of the facial bones. To investigate a possible function of Trps1 during tooth development, we analyzed Trps1 expression from tooth initiation at embryonic day 11.5 (E11.5) to an advanced stage of crown morphogenesis at post-natal day 0 (P0). At E11.5, when localized thickenings of the oral epithelium mark the future sites of tooth development, Trps1 is expressed in both the epithelial thickenings and the underlying neural-crest-derived mesenchyme (Fig. 1B  ). In the first branchial arch, which will subsequently give rise to the lower jaw, Trps1 mesenchymal expression appears restricted to the oral part of the mandible (Fig. 1A). Subsequently, at the bud (E13.5) and cap (E14.5) stages, Trps1 is strongly expressed in the condensing dental mesenchyme and the future dental follicle (Figs. 1C, 1D). At birth, crown morphogenesis is almost complete, and two specialized cell types—the ameloblasts and odontoblasts—have differentiated, and are responsible for the secretion of enamel and dentin matrices, respectively. At this stage, Trps1 is strongly up-regulated in non-secreting pre-odontoblasts in both molars and incisors (Figs. 1E-1G). Trps1 expression is then down-regulated as soon as pre-odontoblasts fully differentiate in mature odontoblasts and start secreting dentin matrix (Fig. 1H). Strong expression of Trps1 is maintained in the dental follicle (Figs. 1E-1H).
To identify a functional role for Trps1 during tooth development, we analyzed mice carrying a deletion of the GATA Zfn domain of Trps1 (Malik et al., 2002). E18.5 mouse embryos homozygous mutant for Trps1 had no obvious dental abnormalities. They exhibited the expected number of teeth (3 molars and 1 incisor per quadrant) (Fig. 1I  ), and the shapes of molar and incisor crowns appeared to be normal (Fig. 1J). No ectopic budding of the oral epithelium that would be suggestive of the arrested development of supernumerary teeth was observed at earlier stages of tooth development in Trps1 gt/Trps1gt mutants (E12.5 and E13.5). An abnormally arched palate was previously described for both heterozygous and homozygous Trps1 mutants (Malik et al., 2002). We observed a cleft palate in E18.5 homozygous mutants (Fig. 1I), which has not been reported previously.
Mutation in TRPS1 Associated with Supernumerary Teeth
Oral examination showed severe mandibular prognathism (Fig. 3A ) and rounded maxillary premolars and molars, while the mandibular teeth appeared normal. Cephalometric analysis showed a normally positioned maxilla, very prognathic mandible, high mandibular plane angle, increased gonial angle, reduced facial depth length, and long anterior facial height (Fig. 3B). Panoramic radiograph showed 5 supernumerary teeth in the premolar and molar areas of the maxilla and mandible. They all appeared to have the morphology of premolars (Fig. 3C).
Mutation screening of the participant was achieved by PCR amplification and direct sequencing of a DNA sample. A heterozygous 2756C>T mutation was found in exon 6 of TRPS1 (Fig. 3D ). This mutation causes the amino acid substitution alanine to valine at position 919 (A919V). This conservative amino acid change occurs in the zinc-coordinating domain of the GATA-type zinc-finger. This particular alanine is evolutionarily conserved in all mammalian GATA and GATA-like Zfn, suggesting its importance in normal GATA zinc structure and function (Hilton et al., 2002).
In the mouse, expression of Trps1 is found in the mesenchymal cells of the developing tooth germs, first in the dental mesenchyme condensing around the developing teeth, and later in the dental follicle and pre-odontoblasts. Interestingly, Trps1 expression is down-regulated in mature dentin-secreting odontoblasts, suggesting a transient role of Trps1 in odontoblast differentiation. Strong expression of Trps1 in the oral mesenchyme at the time of tooth initiation (E11.5) is consistent with a possible role of Trps1 in early tooth determination. In the mandible, this early expression of Trps1 may also be significant regarding the mandibular prognathism observed in our TRPS individual. We have demonstrated that mice lacking the GATA domain of Trps1 can display a cleft palate, a feature not reported previously. A high-arched palate has been described for several persons with TRPS I (Goodman et al., 1981; Howell and Wynne-Davies, 1986), and one submucous cleft palate was reported in a person with TRPS II (Morioka et al., 1999). Otherwise, Trps1 mutant mice had a normal dental formula, and tooth development proceeded normally. So far, all missense mutations found in the GATA DNA-binding domain of TRPS1 have been associated with the more severe TRPS type III (Lüdecke et al., 2001; Kobayashi et al., 2002). Two different missense mutations at position 919 are among them. The A919T mutation was described in four individuals from three families (Lüdecke et al., 2001) diagnosed as TRPS III. The same mutation as in our patient (A919V) has also been described in a person with TRPS III (case T0202, Hilton et al., 2002). Our patient has a normal stature, but short hands with short metacarpals, which is an intermediate phenotype between TRPS I and TRPS III. In addition, he displays several supernumerary teeth in the molar region and has mandibular prognathism. Unfortunately, the dental status of none of the persons with missense mutations at amino acid position 919 has been reported. And vice versa, none of the rare individuals reported in the literature as having supernumerary teeth (Goodman et al., 1981; Peterson and Thomas, 2000; Karacay et al., 2007) has been molecularly examined. Thus, the identification of the TRPS1 mutation in the present case may help us understand the genetic basis of supernumerary teeth in persons with TRPS. The IKAROS-like zinc-finger of TRPS1, located at the C-terminal end of the protein, is known to mediate protein-protein interactions with other IKAROS isoforms (Sun et al., 1996). Homo- or heterodimerization may therefore be necessary for TRPS1 normal function. The A919V mutation presumably affects the DNA-binding affinity of the GATA zinc-finger, and TRPS1 with a missense mutation may compete with wild-type TRPS1 in a multimeric transcription control complex. Alternatively, the A919V missense mutation may cause a gain of function of TRPS1 and be responsible for the less common TRPS clinical manifestations, such as supernumerary teeth and mandibular prognathism. It is therefore possible that mice lacking the GATA domain of TRPS1 are not a suitable model for addressing the function of TRPS1 during tooth development, since deletion of the GATA domain of mouse Trps1 does not mimic the human missense mutation in the person with supernumerary teeth. The absence of a tooth phenotype in the Trps1 mutant mice is overall consistent with the majority of people with TRPS with non-sense mutations who do not exhibit any obvious dental anomalies. The missense mutation we identified in the person with TRPS with additional dental anomalies is unusual and has been previously reported in only one other person, the dental status of whom was not described. The strong, localized expression of Trps1 during tooth development is suggestive of a role that is clearly not revealed by this particular missense mutation. An amino acid change in an important DNA-binding domain could result in an increase or decrease in binding or alter binding specificity. The rather mild "facial" TRPS features of our participant, together with the unusual tooth phenotype, which suggest a hypomorphic loss of function together with a change in DNA-binding target specificity, might be the consequence of the A-V mutation. It was recently shown that TRPS1 can bind the promoter of RUNX2 and inhibit the activity of the RUNX2 promoter in vitro (Napierala et al., 2005). RUNX2 is a transcription factor that belongs to the runt domain family of proteins. Heterozygous mutations of RUNX2 in humans lead to cleidocranial dysplasia (CCD; OMIM 119600), an autosomal-dominant skeletal dysplasia characterized by short stature, hypoplastic clavicles, large fontanelles, and orofacial abnormalities (Mundlos, 1999). The palate is often highly arched, and cleft palates have been described. Dental abnormalities include delayed eruption of permanent teeth associated with the presence of a very large number of unerupted supernumerary teeth, which appear to be morphologically normal. During mouse tooth development, Runx2 transcripts are found in abundance in the dental mesenchyme at the bud and cap stages. At later stages, Runx2 expression is absent in odontoblasts, but strong in the dental follicle (Yamashiro et al., 2002). Co-expression of Runx2 and TRPS1 in dental mesenchymal cells is consistent with a possible interaction between Runx2 and TRPS1 during early tooth development. It is striking that extra teeth are observed both in persons with CCD and in those with unusual TRPS. If, during tooth development, Trps1 belongs to the same pathway as Runx2 and acts as a repressor of Runx2, it is likely that the TRPS1 mutation A919V results in a gain of function of TRPS1, since the supernumerary teeth observed in our TRPS participant mimic the dental phenotype of persons with RUNX2+/–. In summary, expression of Trps1 during mouse tooth development and the discovery of a TRPS1 missense mutation in a person with TRPS with supernumerary teeth suggest a new role for TRPS1 in tooth morphogenesis. It is crucial to identify the function of mutant A919V TRPS1 if we are to understand the basis of the generation of extra teeth. Further analyses of TRPS1 mutations in other individuals, combining clinical features of TRPS with dental anomalies, is likely to shed new light on TRPS1 function.
We are grateful to the participant and his family for allowing us to use his medical and dental information for publication. The research is supported by The Thailand Research Fund (TRF) to P.K., Research Councils UK (RCUK) to I.M., and the Deutsche Forschungsgemeinschaft (DFG) to H.-J.L.
* authors contributing equally  Received for publication April 23, 2007. Revision received May 29, 2008. Accepted for publication August 1, 2008.
Journal of Dental Research, Vol. 87, No. 11,
1027-1031 (2008)
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ABSTRACT
INTRODUCTION
