hypodontia: genetics and future perspectives

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Braz J Oral Sci. April/June 2005 - Vol. 4 - Number 13

Hypodontia: genetics and futureHypodontia: genetics and futureHypodontia: genetics and futureHypodontia: genetics and futureHypodontia: genetics and futureperspectivesperspectivesperspectivesperspectivesperspectives

TTTTTrevor J Pembertonrevor J Pembertonrevor J Pembertonrevor J Pembertonrevor J Pemberton11111

Parimal DasParimal DasParimal DasParimal DasParimal Das33333

Pragna I PatelPragna I PatelPragna I PatelPragna I PatelPragna I Patel1,21,21,21,21,2

1Institute for Genetic Medicine and the 2Centerfor Craniofacial Molecular Biology, Keck Schoolof Medicine, University of Southern California,CA-USA and 3Department of Surgical Oncology,M.D. Anderson Cancer Center, Houston, TX-USA

Received for publication: February 14, 2005Accepted: May 10, 2005

Correspondence to:Correspondence to:Correspondence to:Correspondence to:Correspondence to:Pragna I PatelInstitute for Genetic Medicine, Keck School ofMedicine, University Of Southern California,2250 Alcazar Street, CSC-240, Los Angeles, CA90033, USA.Tel: +1 (323) 442 2751Fax: +1 (323) 442 2764E-mail: [email protected]

AbstractAbstractAbstractAbstractAbstractTooth development is a complex process of reciprocal interactionsthat we have only recently begun to understand. With the large numberof genes involved in the odontogenic process, the opportunity formutations to disrupt this process is high. Tooth agenesis (hypodontia)is the most common craniofacial malformation with patients missinganywhere from one tooth to their entire dentition. Hypodontia canoccur in association with other developmental anomalies (syndromic)or as an isolated condition (non-syndromic). Recent advances in genetictechniques have allowed us to begin understanding the genetic processesthat underlie the odontogenic process and to identify the mechanismsresponsible for tooth agenesis. Thus far two genes have been identifiedby mutational analysis as the major causes of non-syndromichypodontia; PAX9 and MSX1. Haploinsufficiency of either has beenobserved to cause the more severe forms of hypodontia whilst pointmutations cause hypodontia to varying degrees of severity. With theprevalence of hypodontia having been observed to have increased duringthe 20th century, the future identification and analysis of its geneticbasis is essential to allow us to better treat the condition. The cliniciancan facilitate this process by collaborating with the human geneticistand referring patients/families with familial hypodontia for investigativeresearch.

Key Key Key Key Key WWWWWords:ords:ords:ords:ords:hypodontia, tooth agenesis, PAX9, MSX1, prevalence, mutations

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Braz J Oral Sci. 4(13):695-706 Hypodontia: genetics and future perspectives

IntroductionIntroductionIntroductionIntroductionIntroductionThe smile is a unique facial expression distinct to primates. Themain physical component of the smile is a complete dentitionset comprising four different types of teeth. Vertebratecomparative histology has indicated that the continuedevolution of teeth throughout the emergence of modern man isdue to the increased fitness they have offered us. Our modernlifestyle also has a special attachment to a complete set ofdentition aside from their use as merely mastigatory appendagesfor food. For this reason, naturalists, biologists and dentistshave for a long time been trying to unravel the cause for thecongenital loss of teeth leading to several clinical phenotypessuch as hypodontia, oligodontia and anodontia.While a number of clinical studies have been carried out ondisorders that involve the congenital lack of teeth1-11, untilrecently very little effort has been made to understand thegenetic component responsible for mammalian toothdevelopment. Advancements in molecular biologyapproaches coupled with the now complete human genomesequence12 has allowed a number of putative disease genes/loci associated with the hypodontia/oligodontia phenotypesto be identified6,13-16. Functional studies of these diseasegenes have started to reveal their precise role in toothdevelopment allowing us to better understand their role indisease pathology and the molecular morphogenetic fieldswithin which they function17.The congenital lack of one or more permanent teeth is acommon anomaly in man. By definition, congenitally missingteeth are those that fail to erupt in the oral cavity and remaininvisible in a radiograph, which implies that this is causedby disturbances during the early stages of toothdevelopment. These phenotypes most frequently involvethe second premolars and upper lateral incisors andcommonly lead to mild phenotypes that have no associatedsystemic disorder. The term hypodontia has most frequentlybeen used for describing the phenomenon of congenitallymissing teeth, although a large number of missing teeth isdefined as oligodontia and the complete absence of teeth isdefined as anodontia. Hypodontia and oligodontia are alsoclassified as either nonsyndromic (isolated) or syndromic(associated with their syndromes). A literature survey showsvarious other terminologies describing a reduction in teethnumber; teeth aplasia, congenitally missing teeth, absenceof teeth, agenesis of teeth, and lack of teeth. Although nodistinct definition and classification exists in literature, thefollowing definitions have been widely used in scientificliterature:1. Hypodontia: one of six missing teeth excluding 3rd molar2. Oligodontia: More than six missing teeth excluding 3rd

molar3. Anodontia: Complete absence of teeth

Identifying disease genesIdentifying disease genesIdentifying disease genesIdentifying disease genesIdentifying disease genes

The online catalog of inherited human diseases (OnlineMendelian Inheritance in Man; OMIM18) currently listsalmost 16000 inherited human disease genes. However, themolecular etiology for only about 2000 of these has beendetermined successfully. The cloning of disease genes isthe first step in understanding the detailed molecular basisof the disease that ultimately facilitates the development ofsuitable diagnostic and therapeutic agents. There are mainlytwo different strategies that have been employed foridentifying human disease genes: functional cloning andpositional cloning (Figure I). As the name implies, functionalcloning identifies genes based on the known biochemicalfunction of their encoded proteins. For example, theidentification of the gene responsible for phenylketonuriawas achieved by purifying the mRNA for phenylalaninehydroxylase, the enzyme normally responsible for theoxidation of phenylalanine to tyrosine but absent insufferers, and using this to screen a cDNA library for thecorresponding DNA sequence19 which facilitated theidentification of its chromosomal location20-21. However, inreality for the vast majority of inherited disorders, knowledgeabout their basic biochemical defect is unknown making theuse of functional cloning impossible which has led to asecond strategy, positional cloning.The positional cloning approach employs one or both of thefollowing strategies; (a) analysis of genetic linkage in familieswith a disease and/or (b) identification of a specificchromosomal aberration(s) in the diseased individual.Successful genetic analysis depends on the followingrequirements; (a) identification of large families segregatingthe disease phenotype, (b) a distinctive diagnostic criterionto distinguish the affected individuals, (c) accurateassessment of the individuals of the family for a knownMendelian or complex pattern of segregation and (d) highlypolymorphic DNA markers.In principle, the basis of linkage analysis is to observe thecloseness of two alleles for two different genes on achromosome. The closer the two alleles are to each other,the more likely they are to segregate together during meiosisdue to the reduced chance of a recombinatorial eventoccurring between them. Logically, this principle is appliedto determine the genetic distance of known genetic markers,or “site locators”, from a disease locus by tracing itssegregation pattern in affected families. In figure II, thedisease locus D is closer to the marker locus represented byallele X/x but distant from the locus represented by Y/y. Dueto this, the disease locus co-segregates with the X locuswhereas it shows random segregation pattern with respectto the Y locus. Essentially, good genetic markers are thosewhich are polymorphic and can be easily assayed in genomicDNA isolated either from lymphocytes or buccal cells.The traditional definition of polymorphism is a naturalvariation in nucleotide sequence which is observed in at

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least 1% of a population and is capable of distinguishingthe parental chromosomes which otherwise seem virtuallyidentical in every respect. The degree of polymorphism ofthe genetic marker has direct bearings in scoring thedistinction. The information on segregation of polymorphicmarkers with respect to the disease phenotype is thenanalyzed using statistical programs in order to derive a LODscore. LOD score, or the logarithm to the base 10 of theodds, is defined as the relative probability of the observeddata being the result of true linkage versus the probabilitydue to chance. Conventionally, linkage is consideredestablished if the LOD score at any given recombinationfraction (θ) is equal to or greater than 3, while linkage isexcluded if the LOD score is equal to or less than –2.Statistically, a LOD score of 3 means that it is 103, or 1000times more likely that the observed pattern of segregationof one marker with respect to another marker or diseaselocus occurred because of linkage rather than chance.Multipoint linkage analysis for determining the disease geneposition with respect to a number of genetic markers in thecandidate region is then used to localize the position of thedisease gene between two flanking genetic markers. The successof positional cloning efforts requires very tightly linked markers.An alternative approach to associate gene(s) with a geneticdisorder is to analyze mutations in candidate genes, thosethat have either a proven or speculated association eitherdirectly or indirectly leading to the disease state. With theadvancement of our present day knowledge about diseasepathology and its consequences at both the biochemicaland physiological levels, mapping human disease genes bythe candidate gene approach plays an important roleespecially in excluding the involvement of a gene for a givenphenotype. Using this approach, the association of multiplegrowth factor and growth factor receptor gene(s) withcongenital teeth agenesis has been excluded22.

TTTTTooth Developmentooth Developmentooth Developmentooth Developmentooth DevelopmentIn mammals, tooth development is a complex process withreciprocal interactions between the dental epithelium andmesenchyme involving the shifting of the odontogenicpotential between these tissues (figure III). The first sign oftooth development is the appearance of the primary epithelialband within which the odontogenic process initiates withthe formation of an epithelial bud. Mesechymal cells thendifferentiate around the bud to form the dental papilla, theprecursor of the tooth pulp and dentin-secretingodontoblasts that appear after a few additional soft-tissuephases that include the cap stage leading to the bell stagewhen the enamel-depositing ameloblasts are formed. Thedentinal matrix then forms at the periphery of the dental papilladuring dentinogenesis and subsequently enamel deposition,or amelogenesis, occurs at the dentino-enamel junction aftera few micrometers of dentin has been deposited. Finally,apposition of dentin and enamel gives way to tooth eruptionand function.Transcription factors and signaling molecules, which operateboth intra- and extra-cellularly, are expressed in a spatially-and temporally-restricted pattern in the epithelium andmesenchyme tissues throughout the odontogenic processand guide tooth development (figure III). Tissue-recombination experiments have helped greatly in developingour understanding of the hierarchy and roles of the variousfactors with the odontogenic process23-24.BMP4, a member of the transforming growth factor-β (TGF-β)family, and the transcription factors PAX9 and MSX1,members of the paired-box domain and homeobox domaingene families respectively, are examples of controlling factorsduring the odontogenic process. The odontogenic potential


Fig. II: Fig. II: Fig. II: Fig. II: Fig. II: Methodology of positional and functional cloning of asingle gene defect.

Fig. I: Fig. I: Fig. I: Fig. I: Fig. I: Segregation of loci within a two generation pedigree. Thedisease locus is labelled “N” for normal or “D” for disease with thetwo marker loci marked as X/x or Y/y where the change in caseindicates different alleles at that position. Allele x appears tosegregate with the disease (D) given their close proximity.

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shifts from the epithelium to the dental mesenchymeconcomitantly with BMP4 expression25. Both PAX9 & MSX1are expressed in the dental mesenchyme and their expressionis key to maintaining the odontogenic potential followingthis shift25. PAX9 has been identified as a key controllingfactor during the odontogenic process with its expressionfound specifically at the prospective sites of all teeth priorto there being any morphological signs of odontogenesis25.A general role for MSX1 in the development of ectodermalderivatives has been suggested14 with it strongly expressedin the dental mesenchyme but notably absent from the dentalepithelia during the bud, cap and bell stages of toothdevelopment26. Tooth development in both PAX9- and MSX1-mutant mice is arrested at the bud stage27-28, suggesting theyhave similar, non-redundant roles in signal progression tothe cap stage of tooth development. Interestingly, PAX9and MSX1 have been reported to have an importantregulatory role in the maintenance of BMP4 expression andsignaling25 implying they may also have a role in odontogenic

Fig. III: Fig. III: Fig. III: Fig. III: Fig. III: Known protein factors involved in tooth development. Namesin bold bold bold bold bold indicate that they have only been identified as involved atone stage and names in italics indicate those factors that have beenfound to be involved in signalling from both the epithelium andmesenchyme.

potential shifts.

Clinical features of HypodontiaClinical features of HypodontiaClinical features of HypodontiaClinical features of HypodontiaClinical features of HypodontiaA congenital anomaly affecting the formation of the dentitionthat results in a reduction in the usual number of the humanpermanent dentition (a total of 32 teeth in both jaws) and/orthe deciduous dentition (20 total teeth in both jaws) iscommonly referred to as hypodontia. Recent studies haveshown that the occurrence of hypodontia has increasedduring the 20th century29. 80-85% of hypodontia casesstudied involved the agenesis of just one or two teeth30,indicating that most people afflicted suffer from a mild formof the disease. The tooth most commonly missing is thethird molar (or wisdom tooth) which is absent in as much as20% of the population2,31-34. The second most commonly

missing tooth is reported to be the maxillary lateral incisorby some investigators30,35-36 or the mandibular secondpremolar by others37-38. The lowest incidence of toothagenesis occurs in the lower central and lateral permanentincisors with agenesis of maxillary permanent central incisors,maxillary permanent cuspids and maxillary permanent firstmolars also rare39.Hypodontia affecting the primary dentition is rare (prevalencerate of <0.5%) and has been observed to afflict both sexesequally39-40. It is often followed by hypodontia in the sameregion of the permanent dentition, which itself has aprevalence, excluding third molars (wisdom teeth), rangingbetween 2.3% and 10%41 and a third of sufferers typicallyhave at least one first-degree relative also afflicted40,42. Severehypodontia, also referred to as oligodontia, involves theagenesis of six or more teeth and like hypodontia of theprimary dentition it is rare afflicting approximately 0.5% ofthe population43.Hypodontia has been identified as both non-syndromic,where it is an independent congenital oral trait, or syndromic,where it is acquired as part of a specific disease. It is anassociated finding in at least 49 syndromes listed in the OnlineMendelian Inheritance in Man database18 implying somefactors involved in tooth development have a wider rolewithin the human body. Other anomalies associated withhypodontia include small tooth size (microdontia), large toothsize (macrodontia) and anomalies in tooth shape, mostcommonly tapering or “peg-shaped” teeth40,44.The non-syndromic form of hypodontia can be sporadic orfamilial and it has been most frequently reported as inheritedin an autosomal dominant45-52 (AD) fashion where it displaysphenotypic heterogeneity as measured by the nature of themissing teeth and other alterations in the teeth. However,autosomal recessive53 (AR), X-linked54-56 and polygenic57-60

inheritance has also been reported.

Geographical, population and gender prevalenceGeographical, population and gender prevalenceGeographical, population and gender prevalenceGeographical, population and gender prevalenceGeographical, population and gender prevalenceHypodontiaHypodontiaHypodontiaHypodontiaHypodontiaVariation is seen in the number of teeth found in both theprimary and permanent dentition, although it is less commonin the primary dentition39, 41. Several population studies havebeen carried out in the past to establish the epidemiological,clinical and genetic characteristics and prevalence ofhypodontia both in primary and permanent dentition throughthe collection of the dental history of individuals belongingto several families representing various countries/geographical locations and races. Table I shows thesummarized data representing the prevalence of hypodontiain primary and permanent dentition in various geographicallocations. It is apparent from these studies that the populationprevalence of hypodontia varies with geographical location.In the primary dentition, Japan shows the greatest prevalenceof hypodontia which is almost three times greater than the


next highest, Finland. Great Britain shows the lowestprevalence which is an eighth of that of Japan with theremaining countries in table I also showing low prevalencesbetween 0.4-0.6%. In the permanent dentition, Saudi Arabiashows the lowest prevalence whilst Iceland exhibits thehighest. The greatest differences are found in thepopulations of both Iceland and Sweden which exhibit aprevalence of hypodontia in their permanent dentition thatis 16 or 19 (respectively) times greater than in their primarydentition. Europe has a prevalence of hypodontia in thepermanent dentition that is at least ten times greater thanany of the European component countries have exhibited inthe primary dentition. This pattern is also seen between theprimary dentition of New Zealand and the permanent dentitionof its neighbor country Australia, whereas Japan has aprevalence in its primary dentition that is only a third of thatin the permanent dentition of its neighbor country China.Interestingly, Saudi Arabia exhibits an equal prevalence ofhypodontia in both its primary and permanent dentition. Ittherefore appears that people of Scandinavian decent arethe most susceptible to hypodontia in the permanentdentition whilst those of Asian or Arabic descent are themost susceptible in the primary dentition. There areapparently no published reports of large scale studies ofpopulations in Latin American countries.A number of investigations have attempted to take intoaccount any possible gender preference in tooth agenesis(table II). Several reports mentioned a lower prevalence oftooth agenesis in males30,38,61 with one study reporting a maleto female ratio62 of 2:3 whilst others have failed to confirm

this63. The data in (table II) shows no clear pattern of genderpreference with fluctuations between male and female biaswhere most studies show ratios that are close to parity.

OligodontiaOligodontiaOligodontiaOligodontiaOligodontiaThe congenital lack of more than six permanent teeth(oligodontia; a severe hypodontia) is also quite prevalent inthe population. Studies on different populations have shownvariation in the prevalence of oligodontia with the differencein the frequency of oligodontia between males and femalesfound to be not statistically significant64-65, nor is thedifference in distribution of missing teeth over mandible/maxilla and right/left sides64-65. Collective data from sixdifferent studies does however indicate that the frequencyof oligodontia is lower in males than females and that thefrequency of missing second premolars or upper lateralincisors is higher in congenital oligodontia66.

EtiologyEtiologyEtiologyEtiologyEtiologyBoth genetic and environmental factors have been found tocontribute to the etiology of tooth agenesis with manytheories having been suggested to explain their affects,particularly prior to the intensive genetic studies performedin recent years38,49,67-68.

Environmental FactorsEnvironmental FactorsEnvironmental FactorsEnvironmental FactorsEnvironmental FactorsEnvironmental factors can cause tooth agenesis by a varietyof means69 that can be broadly placed into two groups:invasive and non-invasive. These can act either additivelyor independently to affect the positioning and physical

TTTTTable I:able I:able I:able I:able I: Prevalence of hypodontia in the primary and permanent dentition in different countries/continents.

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development of the tooth.Jaw fractures, surgical procedures, extraction of thepreceding primary tooth and changes in muscle pressurefrom the facial and lingual sides are all examples of invasivefactors that can affect tooth development and positioningleading to tooth agenesis and impaction38,49,65.It has also been shown that developing teeth are irreversiblyaffected by chemotherapy and irradiation in an age- and dose-dependent manner, with the latter having been shown tocause the more severe effects4-5. Thalidomide (N-phthaloylglutamimide) has been reported to causecongenitally missing teeth in children whose mothers took itduring their pregnancy1,70-71. Nutrient deprivation and seriousillness have also been linked to tooth developmentalproblems, although no definite etiological relationship hasbeen found between hypodontia and systemic diseases38,49,endocrine disturbances72 or ectodermal dysplasia73.A developmental relationship has been proposed betweennerve and hard tissues74 with tooth agenesis linked to the

development of the main innervation paths75-76 where it wasnoted that the regions most commonly affected byhypodontia were the last to undergo innervation. Brainstemanomalies have been shown not to affect toothdevelopment77 indicating that it is local rather than globalnerve development that affects tooth agenesis.

Genetic FactorsGenetic FactorsGenetic FactorsGenetic FactorsGenetic FactorsIn the majority of cases, hypodontia has a genetic basis.Tooth agenesis is found more commonly among individualsrelated to hypodontia patients than in the population ingeneral56 identifying it as a genetic disease. An exhaustivestudy on a Swedish family with 685 family members, including171 probands affected with hypodontia, showed thathypodontia involving permanent teeth is primarilydetermined by genetic factor(s)38. The frequency ofhypodontia among races varies30,78 and greater concordanceof hypodontia is apparent in identical twins than non-identical79-80 with no environmental etiology apparent in

TTTTTable II:able II:able II:able II:able II: Studies comparing gender bias of tooth agenesis (excluding third molar) in the permanent dentition in various countries.

nr = not reported

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afflicted individuals.Familial hypodontia is reported to exhibit mainly autosomaldominant inheritance with incomplete penetrance andvariable expressivity49-52. However, an autosomal recessivemode of inheritance for hypodontia has been reported in aPakistani family which mapped to chromosome 16q12.153 andin another report on Finnish patients that are afflicted with aspecific type of hypodontia (Recessive Incisor Hypodontia;RIH), where patients notably lacked both deciduous andpermanent incisors81. It has also been suggested that it canfollow sex-linked42,59,82 (Patel et al., unpublished results) orpolygenic inheritance patterns54-55,57-58.Recently, direct evidence was gathered for the genetic basisof tooth agenesis thanks to the mapping of human diseasegenes using linkage analysis followed by mutation analysis

of positional candidate genes present in the candidateinterval. Using this gene mapping strategy, autosomaldominant hypodontia has been localized to at least threechromosomal loci to date; MSX183, PAX913 and an unknownlocus on chromosome 106. Five mutations have thus farbeen identified within MSX1 and ten within PAX9 (table III)with both genes also having been found to be deleted inseparate studies of familial hypodontia45,84.

MSX1Although one report has excluded the MSX1 gene as thegene responsible for tooth agenesis85, recent research hasidentified MSX1 as the causative gene for some forms ofcongenital teeth agenesis with five mutations having beenidentified within MSX1 thus far (table III). All of these

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TTTTTable III:able III:able III:able III:able III: Identified mutations in MSX1 and PAX9 that have been found in people afflicted with hypodontia. Tooth is consideredabsent if not present in >50% of patients. Mutation nomenclature used as previously described 124 and ∆ is used to denote genedeletion or protein absence.

= tooth present; = tooth absent in both; = tooth absent in either mandibular/maxillary; AD = autosomal dominant.

mutations have been point mutations with two leading to asubstitution mutation within the protein and the remainingthree form a stop codon that prematurely truncates theprotein. Two mutations fall within the N-terminal region priorto the central homeodomain (M61K & S105X) with theremaining three (Q187X, R196P & S202X) all falling withinthe homeodomain itself (figure 4(A)).Of the two substitution mutations, the M61K mutation15 fallsoutside of the homeodomain of MSX1 and how it affects itsfunction remains unknown but it has been proposed that itmay be through the disruption of protein interactions15. TheR196P mutation83 falls within helix-I of the MSX1homeodomain disrupting its stability and functionalactivity86.Of the three premature termination mutations, S105X is theonly mutation to occur prior to the homeodomain of MSX1(figure 4(A)). It was identified in a Dutch family sufferingfrom cleft lip-palate and hypodontia that were found to beheterozygous for the 314C>A nucleotide substitution, whichcreates a stop codon in MSX1 exon 1 truncating the proteinprior to the homeodomain87. The remaining two terminationmutations fall within the central region of the MSX1homeodomain. A 559C>T nucleotide substitution wasidentified in a Flemish family suffering from cleft lip-palateand hypodontia where it forms a stop codon that truncatesthe protein within helix-I of the MSX1 homeodomain (N187X;figure 4(A))88. The S202X mutation, caused by a 605C>Anucleotide substitution, was identified in a patient with Witkopsyndrome who was suffering from hypodontia89 where itgenerates a stop codon in helix-I of the homeodomain region(figure 4(A)) truncating the protein and disrupting its

functional activity86,90.Interestingly, two of the premature termination mutationswere identified in patients afflicted with both oligodontiaand cleft lip-palate (S105X & Q187X) with the third mutationhaving been identified in an individual with Witkop tooth-nail syndrome (S202X). They all suffer from additionalpathologies other than hypodontia which also afflicts thosewith substitution mutations indicating that that whilst the

substitution mutations disrupt MSX1 activity they do notabolish it whereas the truncation mutations appear to abolishMSX1 activity leading to a more severe phenotype. This issupported by another study that identifiedhaploinsufficiency of MSX1 as the cause of severeoligodontia within unrelated Finnish patients who also hadWolf-Hirschhorn syndrome84. However, there is no clearcorrelation between the severity of the hypodontia and theseverity of the effect on the MSX1 protein caused by theidentified missense mutations.

PAX9In contrast to MSX1, both missense and frame-shift mutationsin PAX9 have been associated with hypodontia (table III).Of the seven missense mutations identified to date, one is apremature termination mutation (K114X) and the remainingsix are all residue substitution mutations. Of these lattermutations, only five generate a substitution in the protein(L21P, R26W, R28P, G51S & K91E) with one believed toprevent PAX9 expression (1A>G). Three frame-shiftmutations have been identified, two of which are caused bythe insertion of a single nucleotide (G73fsX316 &V265fsX316) and the other by the deletion of 8 nucleotideswith the insertion of 288 foreign nucleotides (R59fsX177).All but one of these mutations (V265fsX316) falls within theN-terminal paired-domain (Figure 4(B)) with the exceptionfalling in the approximate middle of the C-terminal region.This single nucleotide insertion falls within exon 4 of thePAX9 gene creating a frame-shift at amino acid 264 (Figure4(B)) which leads to premature truncation of the protein91.The other single nucleotide insertion was a guaninenucleotide that extends a series of five guanines to sixcausing a frame-shift between the N- and C-terminal DNAbinding domains of the PAX9 paired-domain (Figure 4(B))abolishing the C-terminal DNA binding domain13. By far themost severe frame-shift mutation is caused by a 288bpinsertion in the paired domain of PAX9 (Figure 4(B)) in placeof eight deleted nucleotides which leads to a frame-shift thatdisrupts the C-terminal DNA binding region of the paired-domain47. Two nucleotide substitutions within the PAX9paired domain were also identified during this study (L21Pand K91E), both of which fall within the DNA binding regionsof the PAX9 paired domain (Figure 4(B)), N- and C-terminalrespectively, which may affect PAX9’s ability to associatewith DNA and thus its transcriptional activity.The only substitution mutation to cause prematuretermination was an A340T switch that creates a stop codon atlysine 114 producing a truncated PAX9 protein that terminatesat the end of the N-terminal DNA binding region of the PAX9Paired-box domain92 (figure 4(B)). The remaining threemissense mutations that lead to a residue substitution in thePAX9 protein were all identified recently. An R26W mutationwas identified in the N-terminal DNA binding region of the

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Fig. 4: Fig. 4: Fig. 4: Fig. 4: Fig. 4: Location of the mutations stated in table III in the MSX1 andPAX9 proteins.

PAX9 paired domain which has been hypothesized to affectits target DNA specificity93. A mutation two residues furtherinto the N-terminal DNA binding region was identified (R28P)and shown to dramatically reduce the DNA binding abilityof PAX994. The final missense mutation (G51S) lies withinthe boundary region between the N- and C-terminal DNAbinding regions of the PAX9 paired domain95.Haploinsufficiency of PAX9 has been reported in two studiesalthough its cause has been by two drastically differentmechanisms. One study identified a nucleotide substitutionin the first position of the ATG start codon that has beenhypothesized to abolish its expression48 whilst anotheridentified a deletion of the entire PAX9 gene45.All but one of the PAX9 mutations appear to disrupt theDNA binding ability of its paired domain, thus reducing itstranscriptional activity, which appears to be the likely causeof the hypodontia phenotype associated with them. It isinteresting to note that most of the PAX9 frame-shift, deletionand missense termination mutations cause hypodontia inboth the permanent and the primary dentition, whereasmissense substitution mutations affect the permanentdentition only.

Locus 10q11.2A study on He-Zhao deficiency, a distinct form of permanentteeth agenesis which is different from other previouslydescribed disorders, in members of a large Chinese kindredhas identified an unknown locus on chromosome 10q11.2using multipoint linkage analysis6.With the increase in prevalence of hypodontia observed overthe 20th century, the identification of its causative factors isessential for providing treatment to those afflicted in thefuture. Modern molecular genetic techniques have allowedus to start to identify the genetic factors responsible fortooth agenesis but more work is required to discover howmalfunctions in these factors disrupt tooth development.The identification of more families afflicted with hypodontiais key to identifying the molecular processes that underliethis pathology and our knowledge of tooth development.More patients/families presenting familial hypodontia needto be referred to the geneticist for investigative research toincrease the known population of mutations affecting thedentition. To achieve this, dental professionals need tocollaborate with human geneticists like ourselves after theidentification of probands presenting familial hypodontia andjoin in our gene discovery efforts.

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsAcknowledgementsWork in the authors’ laboratory was supported by NIH grantDE014102 (to PIP).

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