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OULU 2006

D 885

Raija Lähdesmäki

SEX CHROMOSOMESIN HUMAN TOOTH ROOT GROWTHRADIOGRAPHIC STUDIES ON 47,XYY MALES,46,XY FEMALES, 47,XXY MALES AND45,X/46,XX FEMALES

FACULTY OF MEDICINE, INSTITUTE OF DENTISTRY,DEPARTMENT OF ORAL DEVELOPMENT AND ORTHODONTICS,UNIVERSITY OF OULU

D 885

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TA R

aija Lähdesmäki

D855etukansi.fm Page 1 Thursday, September 7, 2006 9:03 AM

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 8 8 5

RAIJA LÄHDESMÄKI

SEX CHROMOSOMES IN HUMAN TOOTH ROOT GROWTHRadiographic studies on 47,XYY males, 46,XY females, 47,XXY males and 45,X/46,XX females

Academic Dissertation to be presented with the assent ofthe Faculty of Medicine, University of Oulu, for publicdiscussion in Auditorium 1 of the Institute of Dentistry,on September 16th, 2006, at 12 noon

OULUN YLIOPISTO, OULU 2006

Copyright © 2006Acta Univ. Oul. D 885, 2006

Supervised byProfessor Lassi Alvesalo

Reviewed byProfessor Timo PeltomäkiProfessor Janna Waltimo-Sirén

ISBN 951-42-8169-1 (Paperback)ISBN 951-42-8170-5 (PDF) http://herkules.oulu.fi/isbn9514281705/ISSN 0355-3221 (Printed )ISSN 1796-2234 (Online) http://herkules.oulu.fi/issn03553221/

Cover designRaimo Ahonen

OULU UNIVERSITY PRESSOULU 2006

Lähdesmäki, Raija, Sex chromosomes in human tooth root growth. Radiographicstudies on 47,XYY males, 46,XY females, 47,XXY males and 45,X/46,XX femalesFaculty of Medicine, University of Oulu, P.O.Box 5000, FI-90014 University of Oulu, Finland,Institute of Dentistry, Department of Oral Development and Orthodontics, University of Oulu,P.O.Box 5281, FI-90014 University of Oulu, Finland Acta Univ. Oul. D 885, 2006Oulu, Finland

AbstractStudies on families and individuals with sex chromosome abnormalities and 46,XY females, togetherwith molecular research, have provided proof that both X and Y chromosome genes are expressed inhuman tooth crown growth. The Y chromosome promotes the formation of both permanent toothcrown enamel and dentin, whereas the effect of the X chromosome is seen mainly in enamelformation. In particular, the effect of the Y chromosome on dentin formation explains the expressionof sexual dimorphism in crown size. When crown growth is complete, root dentin is formed andrequires proliferation of epithelial cells in Hertwig's epithelial root sheath to initiate thedifferentiation of root odontoblasts. These epithelial cells determine the size, shape and number of theroots. There is a clear sex difference in tooth crown sizes, men have larger teeth than women. The aimof this research was to study completed permanent tooth root lengths in individuals with sexchromosome abnormalities and 46,XY females, an approach which might also provide some clues fora further insight into the development of sexual dimorphism in human growth. The underlyinghypothesis was that the effect of the X and Y chromosomes on crown growth is also expressed in rootgrowth.

The subjects were participants of L. Alvesalo's research project, Kvantti, and comprised 45,X/46,XX females, 47,XYY and 47,XXY males and female sex reversals with insensitivity to androgens(46,XY females). The root lengths were measured from dental panoramic radiographs with a slidingdigital calliper. All available teeth (except third molars) with complete root formation on both sidesof the jaws were measured.

The results showed longer final permanent tooth root lengths in 47,XYY and 47,XXY males,while the roots in 45,X/46,XX females were shorter compared with the values of normal men andwomen, respectively. The root lengths of 46,XY females were longer compared to normal women andplaced on a level with normal men. The root morphology did not reveal any major deviations fromnormal variation. In terms of population dental developmental standards it is conceivable thatchanges in these study groups in final size of their permanent tooth roots become evident during aperiod beginning eight years after birth and continuing up to the age of 14 years, at least.

It became clear that the effect of the Y chromosome on tooth root growth is greater than that ofthe X chromosome, and this may cause the observed sexual dimorphism, males having longer rootsthan females. It is suggested that root growth may be affected by the same genes on the X and Ychromosomes which promote crown growth.

Keywords: aneuploidy, chromosomes human X, chromosomes human Y, growth anddevelopment, humans, sex characteristics, tooth root

Just keep on trusting that everything is much more wonderful

than we can understand, it is the truth. Vincent van Gogh

To my children Perttu, Mauri, Esko, and Seppo

Acknowledgements

This study was carried out at the Department of Oral Development and Orthodontics, Institute of Dentistry, University of Oulu, first during postgraduate studies and later as a part-time job in addition to work as a clinical orthodontist in the Kuopio and Iisalmi district health care centres. The theoretical studies and writing of the summary to the thesis were carried out in the last year as a part of full-time work at Oulu University.

This study is a part of a larger research series belonging to Lassi Alvesalo’s research project, Kvantti, on individuals with sex chromosome abnormalities. The clinical examinations of the patients and relatives, and the documentation were performed by Professor Lassi Alvesalo and his research team, mainly at the Institute of Dentistry, University of Turku, during the 1970s and 1980s. I am most grateful to Professor Lassi Alvesalo, D.Odont., head of the Orthodontic Department, for the opportunity to take part in his research project. My first interest was to find out factors affecting growth in humans, and as an interesting response to this I was asked to study human tooth root growth, which was suggested by Professor Lassi Alvesalo. I want to thank him for the immensely valuable work he has done in conducting me into the world of science in an extremely forbearing and sensible manner.

Special thanks go to Professor Erkki Tammisalo, D.Odont. for his great contribution to the accomplishment of radiographic examinations. This work was based entirely on that radiographic method as carried out by him at the Institute of Dentistry, University of Turku.

The statistical calculations were performed by Ahti Niinimaa, Ph.D., who also kindly advised me on related problems whenever needed. Malcolm Hicks, M.A. (Cantab.) revised the English language of the reports during all stages of this thesis. He has been of great help in finding good and simple phrases to express the complex notions we have sometimes been dealing with.

Sincere thanks to the official reviewers of the thesis, Professor Janna Waltimo-Sirén, D.Odont. Institute of Dentistry, University of Helsinki, and Professor Timo Peltomäki D.Odont., Clinic for Orthodontics and Pediatric Dentistry, Center for Dental and Oral Medicine and Cranio-Maxillofacial Surgery, University of Zürich, Switzerland, for the thorough and constructive comments on the main details and concepts in this thesis. They both get my special thanks for the opportunity to have profound discussions in this

subject and tools to clarify the message. I also want to thank in advance my opponent Professor Grant Townsend, D.Odont., Associate Dean (Academic), Dental School, University of Adelaide, Australia.

I received irreplaceable help from Ms Sirpa Väätäjä on many occasions during these years, while statistician Päivi Laukkanen, M.Sc. helped me to create the computer databases and secretary Ms Eija Tuomi helped me in transferring some of the data to the computer program.

The staff at the Institute of Dentistry has provided good discussions and support during these years, which has greatly encouraged me to continue with the work. The head of the orthodontic department today, Professor Pertti Pirttiniemi, D.Odont., has adopted a very stimulating attitude towards my studies and the personnel of the department have given me most valuable help and support, which has made this all possible. Also I want to thank the Deans of the Institute of Dentistry Professors Aune Raustia, D.Odont. Kyösti Oikarinen, D.Odont. and Hannu Hausen, D.Odont. for providing the facilities to carry out the research.

The Kvantti research project was supported during the earlier years by the Emil Aaltonen Foundation, the University of Turku Foundation and the Academy of Finland. In addition, this study has been supported by the University of Oulu, the Finnish Dental Society, Apollonia and the Orthodontic Section of the Finnish Dental Society, Apollonia.

My parents have been most helpful and supportive to me in my career in so many ways during all these years. They have always had faith in my visions, and I am most grateful for that.

Last but not least, I wish to thank my boys, Perttu, Mauri, Esko and Seppo, for their patience and great understanding during these years when I have been working on this thesis away from home. I hope they feel that it was worthwhile, because it has been so rewarding for me.

Abbreviations

45,X females with one X chromosome (Turner women) 45,X/46,XX a mosaic female in whom a second cell line has the chromosome

constitution 45,X (Turner women) 46,X,i(Xq) an isochromosome female in which a second cell line is monocentric

(two long arms in the X chromosome) (Turner women) 46,XX normal female sex chromosome constitution (a normal woman) 46,XY normal male sex chromosome constitution (a normal man) 46,XY female a female with a male sex chromosome complement

(a female sex reversal) 47,XXY individuals with an extra X or Y chromosome (Klinefelter men) 47,XYY males with an extra Y chromosome AMELX X-linked amelogenin gene AMELY Y-linked amelogenin gene HERS Hertwig’s epithelial root sheath Xp, Yp short arm of the X or Y chromosome Xq, Yq long arm of the X or Y chromosome

List of original articles

I Lähdesmäki R, Alvesalo L (2004) Root lengths in 47,XYY males’ permanent teeth. J Dent Res 83: 771-775.

II Lähdesmäki R, Alvesalo L (2005) Root growth in the teeth of 46,XY females. Arch Oral Biol 50: 947-952.

III Lähdesmäki R, Alvesalo L (2006) Root growth in the permanent teeth of 45,X/46,XX females. Eur J Orthod 28: 339-344.

IV Lähdesmäki R, Alvesalo L. Root growth in the teeth of Klinefelter (47,XXY) men (manuscript).

In the text the articles above are referred to by their Roman numerals.

Contents

Abstract Acknowledgements Abbreviations List of original articles Contents 1 Introduction ................................................................................................................... 15 2 Review of the literature ................................................................................................. 17

2.1 Dental development................................................................................................17 2.1.1 Teeth ................................................................................................................17 2.1.2 Development of the dentition ..........................................................................19 2.1.3 Tooth crown development ...............................................................................20 2.1.4 Tooth root development...................................................................................22 2.1.5 Sexual dimorphism in tooth development .......................................................24 2.1.6 Aberrations in tooth root development ............................................................26

2.2 Human sex chromosomes .......................................................................................28 2.2.1 The genome .....................................................................................................28 2.2.2 Number of human chromosomes.....................................................................28 2.2.3 Dimorphic X and Y chromosomes...................................................................29

2.3 Individuals with abnormal sex chromosome complement and the female sex reversal ............................................................................................................31

2.3.1 45,X/46,XX females or females with shortage of sex chromosome material............................................................................................................31

2.3.2 47,XXY and 47,XYY male-trisomies..............................................................32 2.3.2.1 Body growth and craniofacial characteristics in 47,XXY males ..............33 2.3.2.2 Body growth and craniofacial characteristics in 47,XYY males ..............34

2.3.3 Female sex reversals (46,XY females) ............................................................34 2.4 Tooth growth and an abnormal sex chromosome complement...............................35

3 Aim of this research....................................................................................................... 38 4 Subjects and Methods.................................................................................................... 39

4.1 Population...............................................................................................................39 4.2 Methods ..................................................................................................................41

4.2.1 Dental panoramic tomogram ...........................................................................41 4.2.2 Root length measurement ................................................................................42 4.2.3 Reliability of the measurements ......................................................................44 4.2.4 Statistical analyses...........................................................................................45

5 Results ........................................................................................................................... 46 5.1 Root lengths in the permanent teeth of 47,XYY males (I) .....................................46 5.2 Root lengths in the permanent teeth of 46,XY females (II) ....................................47 5.3 Root lengths in the permanent teeth of 45,X/46,XX females (III)..........................47 5.4 Root lengths in the permanent teeth of 47,XXY males (IV)...................................47

6 Discussion ..................................................................................................................... 50 7 Conclusions ................................................................................................................... 55 References Original articles

1 Introduction

Teeth are vertebrate organs. Their development in humans starts in the sixth week of embryonal life, with epithelial ingrowths into the ectomesenchyme at sites corresponding to the future deciduous teeth. The developing tooth undergoes complex morphogenesis regulated by interactions between the epithelial and mesenchymal tissue layers. Signaling molecules appear to regulate the early steps of tooth morphogenesis, and transcription factors associated with these pathways have been shown to be necessary for tooth development (Thesleff 1995; Thesleff & Sharpe 1997; Dong et al. 2005). Tooth formation is a repetitive process, resulting in two dentitions, deciduous and permanent. Human teeth reach their full size shortly after eruption into the occlusion. Studies on families by Alvesalo (1971) and on individuals with sex chromosome abnormalities by Alvesalo and co-workers in the Kvantti research project (e.g. Alvesalo 1997), together with molecular research (Lau et al. 1989), have provided proof that both X and Y chromosome genes are expressed in tooth crown growth. The Y chromosome promotes growth of the permanent tooth crown enamel and dentin, whereas the effect of the X chromosome seems to be restricted mainly to enamel formation (Alvesalo 1997). Enamel formation is decisively influenced by the cell secretory function of ameloblasts that differentiate from the enamel epithelial cells after dentin formation has started in the dental mesenchyme. The differentiation of dentin forming cells, odontoblasts, is preceded by cell proliferations or mitotic activity in the enamel epithelium. It has been suggested that the effect of the Y chromosome, particularly by increasing cell proliferation, explains the expression of sexual dimorphism in crown size and shape, maturation and the number of teeth, i.e. supernumerary permanent teeth are approximately twice as common in normal males as in normal females and permanent ordinary teeth are more frequently missing in females than in males. Moreover, assuming genetic pleiotropy, the expression of the torus mandibularis, statural growth and the sex ratio (the ratio of the number of males to that of females) at birth and in the earlier stages of development are explained by this effect of the Y chromosome (Alvesalo 1985; Alvesalo 1997). Unlike bone, teeth grow without constant resorption and remodelling, to form persisting mineralized dental tissues, enamel, dentin and cementum, providing a valuable record of prenatal

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and postnatal growth. The continuous dentin deposition in the pulpal direction does not affect external size or shape of the teeth.

When tooth crown formation is completed, the differentiation of root odontoblasts is induced by a double layer of epithelial cells in Hertwig’s epithelial root sheath. The dividing epithelial cells in the root sheath determine the size, shape and number of the roots. Formation of the primary dentin continues until the external root form is completed. It has been shown that the cells of the inner layer of Hertwig’s epithelial root sheath secrete small amounts of amelogenin during early differentiation of the root odontoblasts, at a time when the adjacent dental papilla cells are still undifferentiated, suggesting a role for amelogenin in the induction of root odontoblast differentiation. There are morphological differences between the fully differentiated odontoblasts in the crown and root, the coronal odontoblasts being columnar and the root odontoblasts cuboidal, and there may also be qualitative and quantitative differences in the inductive mechanisms operating in the crown compared with the root. There is a clear sex difference in tooth root lengths, males having larger teeth than females, and the roots are definitely shorter in 45,X females, or females with a shortage of sex chromosome material compared to normal women.

The aim of this research was to study tooth growth in individuals with sex chromosome abnormalities and 46,XY females with insensitivity to androgens by determining completed permanent tooth root lengths, and gain additional information about the dental development in these individuals, an approach which may also provide some clues for a further insight into the development of sexual dimorphism in human growth. The underlying hypothesis was that the effect of the X and Y chromosomes on crown growth is also expressed in root growth.

2 Review of the literature

2.1 Dental development

2.1.1 Teeth

A tooth is composed of three types of calcified tissue: enamel, dentin and cementum. Dentin forms the bulk of the tooth, the anatomical crown is covered by enamel and the root by cementum. The junction between the enamel and cementum is the cervical margin of the tooth. In addition, a tooth contains a pulp tissue, the sensory and nutritive organ, and is supported by a periodontal ligament. (Fig. 1) Dentin and cementum formation are able to continue throughout life, in the pulpal surface in the case of the dentin and on the external surface in the case of the cementum. The continuous dentin deposition in the pulpal direction does not affect external size or shape of the tooth. Dental enamel is the hardest and most densely calcified tissue in the body, being originally of ectodermal origin, and does not show continuous formation. Enamel consists of approximately 96 percent inorganic material, made up mainly of hydroxyapatite crystals enveloped in traces of organic material. Dentin is approximately 70 percent mineralized with hydroxyapatite crystals (Ten Cate 1994), while the rest of it, the organic component, consists 86 percent of fibrous protein collagen type I. Unlike bone, the teeth grow without constant resorption and remodelling, to form permanent dentin and enamel structures, providing a valuable record of prenatal and postnatal growth.

After the teeth have erupted into the occlusion they are worn down, and thus continue to erupt slowly to maintain contact with the opposing teeth (Osborn 1981). Tooth wear is, however, relatively slight in modern man. Although under genetic control, the teeth are directly influenced by the environment (e.g. Selmer-Olsen 1949). They have significantly reduced in size during human evolution, the molars first, followed by the front teeth, as larger teeth were no longer necessary for survival. The range of variation in tooth crown size is slightly larger in the maxilla than in the mandible (Reddy 1992).

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Fig. 1. Tooth structures

The developing tooth undergoes complex morphogenesis regulated by interactions between the epithelial and mesenchymal tissue layers (Thesleff & Hurmerinta 1981; Ten Cate 1994; Thesleff & Sharpe 1997; Radlanski 2005; Laurikkala et al. 2006; http://www.bite.it.helsinki.fi). Signaling molecules appear to regulate the early steps of tooth morphogenesis, and transcription factors associated with these pathways have been shown to be necessary for tooth development (Thesleff & Sharpe 1997). New evidence has shown changes in the signaling pathways former supposed to be unchanged between the animal species in the form that new signaling parts connect earlier ones (Varjosalo et al. 2006). Msx-1 homeobox gene expression may be the key signaling mediator during tooth morphogenesis (Thesleff 1995; Han et al. 2003). Dlx homeobox gene has also been shown to be critical for tooth development (Dong et al. 2005). A major question in modern biology is how gene mutations and other structural alterations affect development and are translated into evolutionary changes in morphology (Line 2003). The expression of the Myc genes has been shown in mouse to control multiple independent tissue specific enhancer modules, which have implications for organ specific growth control and they have an expression for instance in the tooth bud (Hallikas et al. 2006).

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2.1.2 Development of the dentition

Migratory neural crest cells, once they have transformed into ectomesenchyme, provide much of the connective tissue associated with the face and the facial bones, and also the tissues of the teeth (except for the enamel) and their supporting structures (Ten Cate 1994). During the first four weeks of embryonal life the frontonasal process and the branchial arches are formed starting to form the processes of the facial structures. By four weeks some of the epithelium is already forming teeth, but not until a week later in the premaxillary area. The positions of the neural crest cells in the maxillary and mandibular processes are associated with their original positions in the mid-brain region and with the time when the cells leave the crest. Specific combinations of homeobox containing genes expressed in the neural crest cells may regulate the types of teeth and their patterning (Thesleff & Sharpe 1997). It is known that the migratory pattern of neural crest cells is not intrinsic but is determined by directory factors in the host tissues. Studies on mice have shown that the specialized dental epithelium directs tooth development prior to the bud stage (Mina & Kollar 1987; Lumsden 1988).

Proliferative activity within the ectoderm derived dental lamina leads to epithelial ingrowths at sites which correspond to the future deciduous teeth, and to an even more active mitotic activity in the underlying ectomesenchyme (Fig. 2a). The tooth buds in human embryos appear in serial order from anterior to posterior part of the forming dental arch, and show considerable variation in their growth rates, on which the order of tooth eruption depends. The germs for each tooth type have the same genetic constitution and potential, but their morphological appearance is dependent on their position in the dentition (Reddy 1992). The ectomesenchyme, once determined, not only induces further development of the tooth but also determines its identity. The alveolar plates form a trough of bone around the developing tooth germs, which eventually become enclosed in bony crypts. Postnatally the teeth have a central role in regulating the extent and direction of growth of the alveolar processes of the jaws (Ten Cate 1994). In the last resort, tooth growth is a prerequisite for the development of normal occlusion. The deciduous dentition is initiated by two months of embryonic development (Nery et al. 1970), and the permanent teeth by the twentieth week in utero (Ten Cate 1994).

Heterodonty, divergent development of the teeth in the dentition, has evolved in many vertebrates, particularly mammals (Butler 1995). The number of teeth has diminished from mesial to distal in the premolars and in the reverse order in the molars. Most mammals have two dentitions (deciduous and permanent). The point where the dental lamina joins the dental organ in a deciduous tooth germ gives rise to the development of the tooth germs for the permanent incisors, canines and premolars (Fig. 2d). The molars of the permanent dentition have no predecessors. When the jaws have grown big enough, the dental lamina burrows posteriorly and gives off epithelial ongrowths with the associated ectomesenchymal response, to form the germs of the first, second and third molars. There are higher correlations in dental maturity within the two tooth groups, that is incisors and first molars versus canines, premolars and second molars (Demirjian & Levesque 1980), and the same grouping can be seen in the timing of tooth emergence.

Abnormalities of development may result for instance in missing teeth or the formation of extra teeth, features which seem to be correlated between the deciduous and

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permanent dentitions in the same individual (Grahnen & Granath 1961). Most studies indicate that missing and supernumerary teeth show dominant autosomal transmission (Grahnen 1956; Alvesalo & Portin 1969), whereas others have proposed a complex of genetic factors (Brook 1984). In any case, supernumerary permanent teeth are found approximately twice as often in normal men as in normal women, and ordinary teeth are missing more frequently in women compared to normal men, suggesting sex chromosome effect in the determination of tooth number (Alvesalo 1985; Alvesalo 1997).

2.1.3 Tooth crown development

Tooth crown development proceeds in bud, cap, bell and crown stages, followed by root development (Fig. 2). The epithelial ingrowth during the bud stage is called the dental organ, the one that forms the enamel of the tooth (Fig. 2b). The ball of condensed ectomesenchymal cells, called the dental papilla, located just beneath the dental organ, forms the dentin and pulp of the tooth, and the condensed ectomesenchyme limiting the dental papilla and encapsulating the dental organ, the dental follicle, gives rise to the cementum and supporting tissues of the tooth. In this stage of tooth development, the cap stage, the dental tissues can be identified as discrete entities, comprising a tooth germ (Fig. 2c). (Ten Cate 1994)

Fig. 2. The different stages of tooth crown development starting from the thickened oral epithelium with active mitotic activity in the underlying ectomesenchyme (a), the tooth development proceeds to the bud stage, the dental organ (b), after that comes the cup stage and the developing tooth can be called a tooth germ (c). In the bell stage the outer and inner enamel epithelium are formed and connected in the cervical loop, also, the permanent tooth bud can be seen arising from the dental lamina (d). The formation of the hard tissues, dentin followed by enamel, starts the crown stage (e). The last ameloblasts are lost when the tooth erupts into the oral cavity, the root formation continues until tooth reaches the occlusion (f).

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Through histodifferentiation and morphodifferentiation in the bell stage, similar epithelial cells in the dental organ are transformed into morphologically and functionally distinct components (Fig. 2d). The centre of the dental organ is termed the stellate reticulum. The cells at the periphery of the dental organ form the outer dental epithelium, the cells adjacent to the dental papilla form the inner dental epithelium, and the junctional zone is known as the cervical loop. The folding of the inner enamel epithelium results from its intrinsic growth caused by differential rates of mitotic cell division, which first occurs throughout the epithelium and precedes dentin formation. A structure known as the basal lamina supports the inner dental epithelium and separates it from the mesenchyme.

This is largely the product of epithelial cells, and contributes to epithelial-mesenchymal communication by controlling the passage of molecules between the two tissues. For example, the bone morphogenetic protein synthesized by the inner dental epithelial cells may be secreted into the dental papilla and induce its mesenchymal cells to differentiate into odontoblasts. (Ten Cate 1994)

Differentiation of the odontoblasts starts beneath the enamel knot in the mesial part of the tooth germ, representing the site of the future cusp development, and progresses distally (Kraus & Jordan 1965; Jernvall et al. 1994). It has been shown that expression of the Msx-2 transcription factor gene (MacKenzie et al. 1992) and cytokine fibroblast growth factor 4 (Niswander & Martin 1992) is confined to the enamel knot, which indicates a role for these in determining the pattern of the tooth formation (MacKenzie et al. 1992).

Dentinogenesis, which always precedes enamel formation, marks the onset of the crown stage (Fig. 2e). The odontoblasts begin to elaborate the organic matrix of dentin, and move towards the centre of the dental papilla, establishing the tubular character of the dentin. Dentin mainly consists of type I collagen (86 percent) but it also contains the precursor protein DSPP (DSPP gene mapped to 4q21) for two unique proteins, dentin phosphoprotein and dentin sialoprotein (Butler 1998). Dentin phosphoprotein plays an initiative and regulatory role in the formation of apatite crystals (Butler et al. 2003). As crown development continues, there is a progressive maturation of the cells of the inner dental epithelium down the cusp slopes. It has been suggested that small proteins resulting from deletion of a major part of the amelogenin, the main protein in enamel matrix, may play a signal transduction role that inhibits ameloblast maturation until a layer of mineralized dentin has been formed (Veis 2003). The cells of the inner enamel epithelium differentiate further to preameloblasts, assuming a secretory function and producing an organic matrix against the newly formed dentinal surface. During amelogenesis the enamel-forming cells, or ameloblasts, move away from the dentin, leaving behind an increasing thickness of enamel. Ameloblasts undergo gradually apoptosis following the secretory stage or during maturation (Smith & Warshawsky 1977), the remaining cells are lost as the tooth erupts into the oral cavity (Fig. 2f). The extracellular organic matrix of the enamel is composed mainly of abundant amelogenins (over 90 percent), which are generally hydrophobic in nature, and of acidic proteins originally termed enamelins. Amelogenin is involved in the control of mineral crystal size, morphology and orientation (Gibson et al. 2001).

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2.1.4 Tooth root development

The epithelial and mesenchymal interactions regulate the morphogenesis of normal root development. When crown growth is completed a double layer of cells known as Hertwig’s epithelial root sheath is formed by cells of the outer and inner dental epithelium (Fig. 3). The formation of the root dentin requires proliferation of epithelial cells in Hertwig’s epithelial root sheath to initiate the differentiation of root odontoblasts and determine the size, shape and number of the roots (Ten Cate 1996). It is even likely that there is a linkage between crown shape and root number. As the inner epithelial cells of the root sheath enclose more and more of the expanding dental papilla, they initiate differentiation of the odontoblasts from the mesenchymal cells at the periphery of the dental papilla. Formation of the primary physiological dentin continues until the external root form is completed (Ten Cate 1994). In this way a single-rooted tooth is formed, while in the formation of multi-rooted teeth the primary apical foramen is converted into two or more secondary apical foramina and the tongues of the epithelium grow towards each other.

Fig. 3. The formation of the hard tissues during tooth root development.

The cells of the inner layer of the epithelial diaphragm of the apical end of Hertwig’s epithelial root sheath secrete small amounts of amelogenin during the early differentiation of human root odontoblasts, at a time when the adjacent dental papilla cells are still

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undifferentiated, suggesting a role in the induction of root odontoblast differentiation (Hammarström 1997). The peripheral dentin in the crown contains highly branched dentinal tubules, whereas that in the root is atubular, and only after a certain amount of root dentin is present do the tubules form, somewhat chaotically, what is known as the granular layer of Tomes (Weber 1983). There are morphological differences between the fully differentiated odontoblasts in the crown and root, as the coronal odontoblasts are columnar and the root odontoblasts cuboidal (Avery 1986). There may be both qualitative and quantitative differences in the inductive mechanisms operating in crown and root dentin formation (Thomas 1995). The first–formed radicular mantle dentin is not deposited directly against the basal lamina of the root sheath, while a hyaline layer, formed from an admixture of the connective tissue extracellular matrix and an epithelial secretory product, separates them. The hyaline layer is involved in establishing an initial attachment of the cellular extrinsic fibre cementum to the tooth surface, and must therefore be regarded as a tooth support tissue (Ten Cate 1996). There is increasing evidence that the cells of Hertwig’s epithelial root sheath are involved in the development of both acellular and cellular cementum (Hammarström et al. 1996). The cells of Hertwig’s epithelial root sheath fragment when root dentinogenesis starts, and are known as the epithelial cell rests of Malassez. It has been suggested that these may be necessary for the secretion of non-collagenous proteins – osteopontin and bone sialoprotein – for the formation of cementum (Janones et al. 2005). (Fig. 3)

Taurodontism is a morphological tooth variation in a multi-rooted tooth in which the pulp chamber is long and bifurcates into short root canals near the apical third of the root. It is also a familial trait frequently associated with a variety of systemic disturbances (Jaspers & Witkop 1980). Normal populations have been shown to exhibit taurodontism at frequencies of 0.5-5 percent (Jaspers & Witkop 1980; Shifman & Chanannel 1978). The affected teeth are molars, sporadically lower premolars (Madeira et al. 1986) and occasionally lower canines or upper premolars, in patients with an otherwise normal dentition. Taurodontism has been classically divided by degree into hypo-, meso-, and hypertaurodontism (Shaw 1928) and a pyramidal (cuneiform) tooth displaying a single-rooted wedge shape (Bell et al. 1989). Taurodontism is found in a higher frequency in populations of early man living under relatively primitive conditions. The prevalence of taurodontism increases with additional X chromosomes (Stewart 1974; Jaspers & Witkop 1980; Varrela & Alvesalo 1989), and tooth agenesis, and a certain type of amelogenesis imperfecta are features associated with it. Almost third of all patients with oligodontia have been reported to exhibit taurodontism and reduced tooth lengths (Schalk-van der Weide & Steen 1993), and these features may all be interrelated, resulting from decreased mitotic activity in the cells of the developing tooth germs (Bell et al. 1989).

Little is known about the genetic influences of signaling pathways during the morphogenesis of normal root development. One of the four genes encoding nuclear factor I (NFI) transcription-replication proteins is Nfic. It is expressed in many organs, including the developing tooth. The most prominent phenotypic feature of the mutation of the Nfic gene in mouse is the failure of the differentiation of odontoblasts and/or dentin formation in the lingual dentinal aspect of the incisor root as well as at the base of the molar leading to arrested molar root growth (Steele-Perkins et al. 2003). MT1-MMP is a membrane type matrix metalloproteinase and essential for remodeling of soft and hard

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tissue interfaces. Tooth eruption and root elongation are severely inhibited in mice with deficient MT1-matrix metalloproteinase enzyme (Beertsen et al. 2002).

Soon after root formation is initiated, the tooth begins to erupt, until it assumes its position in the occlusion (Fig. 2f). After that, posteruptive tooth movement accommodates jaw growth and interproximal tooth wear, and compensates occlusal wear of the teeth in relation to the occlusion (Ten Cate 1994). There are differences in root stage and the timing of eruption. Teeth usually emerge when the root has reached three-quarters of its length, except for the upper central incisors and first molars, which emerge with their roots half-formed, and the canines, whose root length is almost complete (Haavikko 1970). The permanent tooth roots apart from those of the third molars complete their growth during a time period beginning at the age of eight years and continuing up to fourteen years, at least (Table 1).

Table 1. Permanent tooth formation stages in years by Haavikko (1970).

Boys Girls Tooth stage Maxilla Mandible

Maxilla Mandible

Central incisor Crc 3.3 - 3.3 - Ac 9.8 8.0 9.3 8.0

Lateral incisor Crc 4.6 3.3 4.4 - Ac 10.8 9.6 9.6 9.0

Canine Crc 4.6 4.3 4.5 4.1 Ac 13.6 13.2 12.7 11.5

First premolar Crc 6.8 5.9 6.3 5.4 Ac 13.3 12.8 12.6 12.1

Second premolar Crc 7.1 7.0 6.6 6.4 Ac 14.0 13.8 13.4 12.8

First molar Crc 3.6 3.5 3.5 3.5 Ac 9.8 9.8 9.2 9.2

Second molar Crc 7.3 7.4 6.9 7.0 Ac 16.2 15.7 15.1 14.7

Crc = tooth crown growth is completed. Ac = tooth root growth is completed and apex closed.

2.1.5 Sexual dimorphism in tooth development

Sexual dimorphism in final form can already be observed some months after birth in deciduous tooth crown sizes (Kari et al. 1980; Harila et al. 2003). In permanent tooth crowns the size difference for the most of the teeth ranges between two to four percent,

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men having larger teeth than women (Selmer-Olsen 1949; Garn et al. 1964; Alvesalo 1971). The proportion differs between the tooth groups, however, being even nine percent in the mandibular canines. By comparison, sexual dimorphism in stature is about three percent in late childhood, rising to approximately nine percent at the end of the pubertal growth spurt (Garn et al. 1966). The size difference between normal men and women in the tooth crowns is solely due to a thicker dentin measured on a radiograph as a distance between mesial and distal dentino-enamel junctions (Alvesalo & Tammisalo 1981; Stroud et al. 1994; Harris & Hicks 1998). Normal men have longer tooth roots than women (Selmer-Olsen 1949), the average difference being six percent in the mandibular canines, premolars and molars (Garn et al. 1978). There also seems to be a clear sex difference in extreme root lengths, with extremely short roots to be found most often in girls and extremely long roots in boys (Jakobsson & Lind 1973).

The sex differences in the mesio-distal diameters of the tooth crowns in deciduous and permanent molars increase from earlier developing teeth to later developing teeth, except that the third molars show the least dimorphism (Kondo & Townsend 2004). Mayhall and Alvesalo (1995) found sexual dimorphism between the earlier developing cusps of the tooth, but not between the last cusps. On the other hand, the opposite has been suspected, in that the first developing cusp, the paracone, may express the least dimorphism (Kondo et al. 2005). The expression of the cusp of Carabelli in the permanent first upper molars shows a large variation, but the dichotomy of having a cusp or not may have a genetic basis (Alvesalo et al. 1975b). Alvesalo et al. (1975b) found no sexual dimorphism in the occurrence or in the degree of expression of Carabelli trait in permanent upper first molars. However, a tendency for greater expressivity of the trait in normal men compared to women has been found.

It has been concluded from the results for sibling and cousin pairs that genes affecting tooth size are apparently situated both on the X and Y chromosome, with different influences on phenotypic quantity so that the observed sexual dimorphism in average tooth crown size is the influence of the Y chromosome genes (Alvesalo 1971). According to studies on individuals with sex chromosome abnormalities by Alvesalo (1997) and co-workers in the Kvantti research project the Y chromosome promotes growth of the permanent tooth crown, both enamel and dentin, whereas the effect of the X chromosome seems to be restricted mainly to enamel formation. In terms of population developmental standards (Haavikko 1970), the size difference in permanent tooth crowns between normal men and women indicates obvious increases in final growth between the time period of two months after birth up to the age of eight years, apparently expressing a continuous gene effect (Alvesalo 1997).

It has been proposed that the genes on the Y chromosome are responsible for the sexual dimorphism in the rate of bone development (Tanner et al. 1959). Girls are generally more advanced in their somatic growth and development than boys up to the pre-adolescent years (Tanner 1978; Garn & Rohmann 1962). The sex differences in tooth development and size manifest themselves before the adolescent growth spurt, indicating that the mechanisms involved are independent of somatic and/or sexual maturity (Demirjian et al. 1985; Kari 1993). There is no sex difference in dental mineralization during the first stages of crown formation in the majority of teeth, but girls are more advanced at the completion of crown development than boys. The difference between the sexes for all teeth in root development is 0.54 years, the largest difference, 0.90 years,

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being seen in the canines (Demirjian & Levesque 1980), being similar to the delay in skeletal maturation in boys (Tanner 1978). Also Kari (1993) has found a clear sex difference in the mineralization stages of the tooth crowns, girls showing a more advanced crown development than boys.

The lower face height, which is connected to the chewing apparatus, shows a greater difference between the sexes than do the other craniofacial dimensions. There seems to be some connection between root length and facial height, focused on the upper first incisors and canines (Selmer-Olsen 1949). The sex differences in the time required for root formation are much less for the second molar than for other teeth (Thompson et al. 1975). The differences in the duration of tooth mineralization between specific teeth are due to delay rather than an increase in the velocity of growth and rates of eruption show only a modest variation. So, the larger teeth in males compared to females erupt later due to the longer mineralization period (Leigh et al. 2005). As an example, the completion of the crowns of human permanent incisors takes three to five years, but the clearly larger permanent incisors in the great apes takes over five years. These facts tie together tooth size and the time required for tooth crown formation (Dean & Beynon 1991).

2.1.6 Aberrations in tooth root development

Abnormal tooth root length and/or morphology have been associated with a number of genetic defects. Hypodontia has been linked to decreased tooth size in the developing teeth of the same dentition (Brook 1984), and dental development has been found to be delayed relative to controls (Uslenghi et al. 2006). Enamel hypoplasia and taurodontism have also been reported to be associated with hypodontia (Lai & Seow 1989).

Cleidocranial dysplasia is an autosomal dominant generalized skeletal dysplasia caused by mutation in the Runx 2 transcription factor gene (Lee et al. 1997; Kim et al. 2006). It is characterized by moderate short stature, delayed skeletal and dental development, numerous supernumerary teeth and non-eruption of permanent teeth, and occasionally excess root development in the first molars (Seow & Hertzberg 1995).

In Down syndrome (trisomy 21) tooth crown and root size is reduced in most cases, with fluctuating asymmetry, and the mesio-distal crown dimensions of the lower incisors are relatively greater than the labio-lingual dimensions, which is the reverse of the normal situation (Townsend & Brown 1983). Frequencies of hypodontia and taurodontism are increased; taurodontic molars have been found in about half of the Down syndrome individuals examined (Rajić & Rajić Meštrović 1998).

A short root anomaly is expressed in the upper incisors and upper and lower premolars, and inherited in an autosomal dominant manner with incomplete penetrance, but its genetic background is unknown (Apajalahti 1999). A general reduction in permanent tooth root lengths and crown size is seen in individuals with a lack of sex chromosome material. That is evident in Turner females, as compared with normal women (Filipsson et al.1965; Alvesalo & Tammisalo 1981; Midtbø & Halse 1994b).

Gene defects that cause abnormalities in tooth structure may also affect the root morphology, involving pulp morphology in the case of enamel defects and root size and pulp morphology in the case of dentin defects. Amelogenesis imperfecta (AI) is an inherited disorder affecting the tooth enamel, usually in both dentitions. Fourteen different subtypes, based upon mode of inheritance and clinical manifestations, have been

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reported (Witkop 1967; Witkop & Sauk 1976). Thus far, mutations causing different types of AI have been identified in genes coding for certain enamel extracellular matrix proteins (amelogenin and enamelin) and proteolytic enzymes required for enamel maturation (MMP-20 and kallikrein-4; reviewed by Stephanopoulos et al. 2005). The AI hypoplastic-hypomaturation form is inherited in an autosomal dominant manner (ADAI), and associated with reduced tooth crown size and enamel thickness, and with enlarged pulp chambers and taurodontism in the molars of some affected individuals. It is allelic with trichodento-osseus syndrome which is caused by mutations in the DLX-3 transcription factor gene (Dong et al., 2005). Taurodontism, pulpal calcifications, coronal defects and unerupted teeth have also presented in some individuals with X-linked AI (Lykogeorgos et al. 2003), resulting from mutated amelogenin gene. In a hypoplastic form of the dominantly inherited form of X-linked AI (XDAI) the entire enamel in men is thin, shiny and yellow-brown, while in women it can be of almost normal thickness with defective vertical ridging and a wide range of individual variation in its manifestation (Greene et al. 2002).

The diseases mainly affecting dentin formation, dentinogenesis imperfecta (DI) and dentin dysplasia (DD) are inherited in an autosomal dominant manner (Witkop 1957). DI types II and III and DD type II are allelic disorders, in which the formation of dentin is affected by mutations in the dentin sialophosphoprotein gene (DSPP, 4q21.3). These three non-syndromic heritable dentin defects can nowadays also be considered to be manifestations of a single disease with milder and more severe forms (Beattie et al. 2006). DI-II, known as a hereditary opalescent dentin condition appears in both dentitions. Markedly short roots and obliterated pulp chambers and root canals are typical. The penetrance is almost complete and expressivity consistent within each family (Shields et al. 1973). Dentin dysplasia (DD) affects both dentitions as well. In DD-II, or coronal DD (Shields et al. 1973), the deciduous teeth are grey or brown in colour and translucent, with their pulp chambers obliterated. The permanent teeth have normal crown and root morphology and colour, but exhibit large coronal pulp chambers with numerous pulpal calcifications (Melnick et al. 1982; Brenneise et al. 1999).

Of genetically unknown origin is still, however, DD-I (“rootless teeth”), in which the tooth crowns are of normal shape, occasionally slightly amber in colour, but the roots are short and conical with an apical constriction and the dentin is dysplastic and has a high radiodensity (Melnick et al. 1982). Pulpal obliteration in the permanent teeth may result in a “half-moon”-shaped pulp chamber and completely obliterated root canals in both dentitions, while dental eruption is delayed (Kosinski et al. 1999; Kalk et al. 1998).

Since type I collagen is the major extracellular component of dentin matrix, it is conceivable that mutations in the two genes that encode type I collagen alpha chains (COL1A1 and COL1A2) also affect dentin. These patients generally exhibit a connective tissue disorder osteogenesis imperfecta, characterized by bone fragility and deformity, blue sclerae, and hearing impairment (Sillence et al. 1979). The affected teeth markedly resemble those seen in DI patients with bulbous crowns, short and blunted roots, constricted coronal-radicular junctions and obliterated pulp chambers and root canals. The pulp chambers may also be abnormally wide prior to the onset of obliteration (Lukinmaa et al. 1987). The deciduous dentition is more affected than the permanent teeth. Teeth are brown-bluish in colour, translucent and prone to breakage. The variability in collagen composition is large, but the first dentin to form is normal (Shields et al. 1973; Waltimo et al.1994).

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Hypophosphataemic vitamin D-resistant rickets is inherited in an X-linked dominant manner, and men are more severely affected. Patients are short in stature, and tooth eruption is delayed, pulp chambers are large and the dentin is abnormal and presents marked fissures and cracks (Melnick et al. 1982).

Hypophosphatasia is a rare inherited metabolic disease of decreased tissue non-specific alkaline phosphatase and defective bone mineralization. A mutation in the gene ALPL (1p36.1-34) is believed to be the cause of this desease. It has clinically five categories. The perinatal and infantile forms are inherited by an autosomal recessive mode, and the childhood, adult, and odontohypophosphatasia forms are inherited in an autosomal recessive and dominant mode. The odontohypophosphatasia form affects mostly uniradicular teeth in both dentitions with abnormal dentin. In these patients teeth are lost due to decrease in cementum formation, pulp chambers are large, and the calcification of the root apices is delayed. Patients with childhood and adult forms exhibit premature loss of deciduous teeth due to lack of cementum formation. (Melnick et.al. 1982; Anadiotis 2003)

2.2 Human sex chromosomes

2.2.1 The genome

Present view of the magnitude of the human genome is between 20 000 and 25 000 genes. Only one to two percent of the genetic material, known as exons, code for or adjust proteins, while the introns, constituting 24 percent of the total genome, include many repeat sequences of the genes, the intergene space being 75 percent. Genetic variation is large, because one gene may produce several gene products, and individual variation is even larger, because exons are randomly read. The main gene structures and functions of 60 000 proteins are known so far. (Frilander 2003)

2.2.2 Number of human chromosomes

Chromosome mutations also include changes in the number of normal chromosomes, as opposed to the number of sets of chromosomes, leading to unequal numbers of different chromosomes in one individual, and this is called aneuploidy. The human somatic cell contains 46 chromosomes: two sex chromosomes and 22 paired autosomes, with one chromosome derived from the mother and one from the father. The X chromosome is comparable in size to autosomes, while the Y chromosome is much shorter. All viable individuals possess at least one X chromosome. Normal women have a 46,XX chromosome constitution and men a 46,XY constitution. The number of autosomes is quite constant, as triploidy and tetraploidy are seldom found and haploidy only in the extremely rare 21 monosomy. Individuals with that kind of chromosome complement are seldom viable because of a serious illness. Aneuploidies for autosomal chromosome 13, 18 and 21 trisomies are found, however. It should be noted that sex chromosomes show a

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much wider range of aneuploidy and the phenotype of these individuals is not so destructive as those for autosomes, mosaicism also is much more common and often involves structurally abnormal X and Y chromosomes. If the trisomic and monosomic cells arising in somatic tissues through the misdivision of the chromosomes or chromosome loss are viable, the result is mosaicism, which means the presence of differing cell lines in relation to genotype in one individual. If the mosaicism occurs very early in development, the entire individual may be mosaic. Patients with more than three sex chromosomes suffer from mental retardation and display various other somatic anomalies. The abnormalities vary considerably from case to case. (Therman & Susman 1993)

Aneuploidy results from non-disjunction in either the meiotic division of the parents or the early cleavage divisions of the affected individuals. Aneuploidy for more than one chromosome is the result of non-disjunction in both meiotic divisions, non-disjunction in the meiosis of both parents, multiple non-disjunction in the same mitosis or meiosis, or abnormalities in more than one mitosis. The incidence of 47,XXX and 47,XXY children increases with maternal age, as does that of autosomal trisomies, indicating non-disjunction in maternal meiosis (Barr et al. 1969; Therman 1986). The incidence of 45,X children seems to be independent of maternal age, however, and studies on the Xg blood group gene have shown that 78 percent of the gamete without another sex chromosome comes from the father (Therman & Susman 1993). Nielsen and Wohlert (1991) found in liveborn children in Århus, Denmark total incidence of sex chromosome abnormalities to be one in 448 children.

2.2.3 Dimorphic X and Y chromosomes

Human sex chromosomes are thought to be descended from a homologous pair of autosomes (Ohno 1967). There is evidence for gene loss from the Y chromosome during mammalian evolution, and mutations and inversions have further reduced the level of homology. The length of the Y chromosome is highly inheritable, the variation affecting mostly the long arm (McKenzie et al. 1972). A regulatory role of the Y chromosome in human growth has been pointed out, especially in the differential ontogenesis of the sexes (Ounsted & Taylor 1972). The morphology of the X chromosome, on the other hand, has changed remarkably little and is still the same between monkeys and man. The gene content in the X chromosome has remained the same throughout mammalian development (Ohno 1967). The X chromosome contains 3 000 genes and the Y chromosome close to hundred.

The double dosage of X chromosomes in women compared with men is compensated for by random inactivation of one X chromosome in each somatic cell at an early stage of embryonic development (Lyon 1962), and the same chromosome remains inactive in all descendants of any given cell. Particularly in humans, several genes are known to escape X inactivation and are expressed in both the active and inactive X chromosomes. These represent about ten percent of the X-linked transcripts (Carrel et al. 1999). For instance the Xg blood group genes, the loci for the blood-group antigens (Ducos et al. 1971), and the X-linked amelogenin gene (AMELX) are located in the short arm of X (Xp)

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chromosome in its always active region (Therman & Susman 1993). X chromosomes in women can change genetic material in recombination during meiosis in the same way as autosomes, whereas recombination of the X and Y chromosomes in men is limited to the pseudoautosomal regions found in the telomeric parts (Schaffner 2004). A dozen pseudoautosomal genes have been identified, most of them on the short arm (Cooper 1999). The male-specific region of the Y chromosome (MSY) is between the pseudoautosomal regions, and comprises 95 percent of the chromosome length. MSY consists of a mosaic of heterochromatic sequences and three classes of euchromatic sequences. Skaletsky et al. (2003) report 78 protein-coding genes in MSY that collectively encode 27 distinct proteins locating in euchromatic sequences. The occurrence of MSY gene pairs subject to frequent conversion might provide a mechanism for conserving gene functions in the course of evolutionary time in the absence of crossing over in the Y chromosome.

X- and Y-linked gene pairs encode very similar but not identical protein isoforms. The coding regions of the X and Y chromosome amelogenin genes, for instance, are approximately 87 percent homologous (Chen et al. 1998) and both of them are transcriptionally active (Lau et al. 1989; Nakahori et al. 1991; Salido et al. 1992; Gibson 1999). Molecular studies have indicated that loci for human amelogenin, the main protein component of the enamel organic matrix, are to be found on the distal short arm of the X chromosome (Xp22.1-p22.3) and possibly on the proximal long arm of the Y chromosome (Yq11), although the short arm of the Y chromosome has also been suggested (Lau et al. 1989; Nakahori et al. 1991; Salido et al. 1992). Amelogenin genes are expressed only in the developing teeth. The level of Y chromosome locus expression is ten percent of that of the X chromosome (Salido et al. 1992). Some Y chromosome-linked genes have assumed a testis-specific expression pattern, whereas their X chromosome-linked homologues are ubiquitously expressed (Foster & Graves 1994).

A gene known as the short-stature homeobox (SHOX) is situated in the freely recombining region of the human sex chromosomes (Kauppi et al. 2004) and proposed to be the cause of the short stature observed in Turner Syndrome individuals (Lahn et al. 2001). Ellison et al. (1997) reported an osteogenic gene named PHOG, which has been shown to be identical to SHOX, and has been mapped to Xp22 and Yp11.3, which encode proteins with different patterns of expression (Rao et al. 1997), and also are suggested loci for amelogenin.

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2.3 Individuals with abnormal sex chromosome complement and the female sex reversal

2.3.1 45,X/46,XX females or females with shortage of sex chromosome material

An American, Henry Turner, was the first to describe in 1938 a syndrome in women with decreased height and lack of development of the secondary sex characteristics. Not until twenty years later in 1959 it was discovered that women with Turner syndrome lack an X chromosome or part of it, and with it the genes for the development of the ovaries, sex hormone production, physical sex development and height (Nielsen et al. 1991). The incidence of Turner syndrome in Denmark is approximately one in 2000 girls (Nielsen & Wohlert 1991), and approximately half of them are 45,X females having one X chromosome in every cell. The mosaic chromosome constitution 45,X/46,XX consists of both normal XX and one X sex chromosome cell lines. It is found in about 20 percent of Turner females. (Nielsen et al. 1991) It has been estimated that 98 percent of 45,X conceptions end in a spontaneous abortion, and many foetuses who survive also have genital, cardiac, renal and dermal abnormalities (Park et al. 1983). The degree of Turner characteristics in mosaic individuals has been found to reflect the degree of mosaicism (Sarkar & Marimuthu 1983). 46,X,i(Xq) is a subgroup of Turner syndrome with a long-arm isochromosome or one X chromosome with two long arms (Xq), caused by a misdivision, and one normal X chromosome.

Sex chromosomes have an effect on body growth and its timing. With a normal chromosome constitution there is a growth spurt in puberty, and many children also have a mild growth spurt between the ages of five and nine years. The mean height for girls is about 165 cm in western countries and it is reached mostly by the age of 16 years. The absence of X chromosome material affects body growth, however, so that the average height of Turner girls (including mosaics) at birth is a few centimetres less than normal (Park et al. 1983; Nielsen et al. 1991) and their adult stature is clearly shorter compared to normal women, being 146.9 cm in a Finnish study (Varrela et al. 1984a). The adult stature of 45,X/46,XX females is above the average for 45,X females (Park et al. 1983). Turner females have changes in body proportions on the longitudinal axis to a greater extent than in body width: first they are short-legged, while in adulthood the body proportions are like in normal women (Park 1977; Varrela et al. 1984a). Their head dimensions are close to those of normal women with a tendency to a narrower upper face (Varrela et al. 1984a), while the facial dimensions are smaller (Grön et al. 1999). They have a delay in skeletal maturity averaging 2.4 years, which is commonly attributed to the lack of primary oestrogen, since growth hormone levels are normal (Webber et al. 1982; Midtbø & Halse 1992). Dental maturity is accelerated, however, the mean difference being one year (Filipsson et al. 1965; Midtbø & Halse 1992). Turner girls are often offered growth hormone therapy from the age of six years and small doses of oxandrolon (a synthetic growth hormone which resembles male sex hormone) when they are 10 years of age, enabling their final height to be increased by 5-10 cm (Nielsen et al. 1991). With

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oxandrolone or growth hormone alone the final height can be increased by 3-4 cm (Naeraa et al. 1990). They are also given oestrogens together with progestogens to ensure normal pubertal development, and this is continued at least until they are in their fifties. As the phenotype of 45,X/46,XX females is in proportion to the rate of mosaicism, they have at least partial natural hormone production (Park et al. 1983; Nielsen et al. 1991) and the great majority of known pregnancies among Turner women involve mosaics.

The facial skeleton in normal individuals is one fourth and the neurocranium is half of the adult size at birth. Thus it is no wonder that the lack of sex chromosome material in Turner females leads to more pronounced growth changes in the facial region than in other head dimensions after birth. The anterior and posterior cranial bases are shorter than normal and flattened (Midtbø et al. 1996; Grön et al. 1999), which may originate from the foetal period (Filipsson et al. 1965; Midtbø et al. 1996), when the primary cartilages form the craniofacial skeleton. The face is retrognathic, i.e. maxillary growth is normal but the mandible is short (Peltomäki et al. 1989; Hass et al. 2001), and the jaws are posteriorly rotated (Midtbø et al. 1996). The occlusal morphology of 45,X females differs from that of normal women in that the prevalences of distal molar occlusion, lateral crossbite, maxillary overjet and tendency for open bite are increased (Laine et al. 1986; Harju et al. 1989; Midtbø & Halse 1996). The dental arch in the mandible is broader and that in the maxilla narrower than in normal women (Laine & Alvesalo 1986). A narrow palate compared to normal women and exostosis on the palatal alveolar plates are frequent features (Laine et. al. 1985). By comparison with the 45,X females, 45,X/46,XX and 46,X,i(Xq) females show a milder expression of malocclusion (Harju et al. 1989). Turner women have been found to have increased asymmetry in tooth size and/or in the morphology of antimeric teeth (Midtbø and Halse 1994a; Pirttiniemi et al. 1998). These findings suggest that another sex chromosome is of importance for harmonious growth and development of the craniofacial skeleton and ultimately normal occlusal morphology and relations (Laine et al. 1986).

2.3.2 47,XXY and 47,XYY male-trisomies

Trisomy of the sex chromosomes, the most prevalent aneuploidy in humans, occurs in three types: XXX, XXY, and XYY. Trisomy has only a minor phenotypic effect, and most of these individuals are never diagnosed (estimated 64 percent of 47,XXY males and 85 percent of 47,XYY males) (Ratcliffe 1999). An American, Harry Klinefelter and associates, were the first to describe in 1942 the syndrome in men with a tendency to breast development, decreased production of sperm cells and increased excretion of pituitary hormones (Nielsen 1999). Individuals with Klinefelter syndrome have two X chromosomes in addition to one Y chromosome (47,XXY), or in rare cases three or four X chromosomes. The chromosome constitution 47,XXY is found in about 80 percent of males with Klinefelter syndrome, six percent of whom have a chromosomal mosaic. Klinefelter syndrome is the most common sex chromosome abnormality, with an incidence of one in 596 newborn boys (Nielsen & Wohlert 1991), although one in 769 has also been suggested (Ratcliffe 1999). 47,XYY males, or males with one extra Y chromosome, represent the only aneuploidy which is not selected against before birth,

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being found in one male birth out of 894 (Nielsen & Wohlert 1991). The chromosome constitution 47,XYY is found in approximately 80 percent of males with an extra Y chromosome.

Normal men and 47,XXY and 47,XYY males have no significant differences in prenatal testosterone levels, while all these groups differ from normal women (Ratcliffe et al. 1994a). Later the hormone levels of 47,XYY males can still be considered normal, although they show greater variability in plasma testosterone levels (BénéZech & Noël 1985), and they usually have normal children. By contrast, the production of testosterone in 47,XXY boys is insufficient later and requires substitution therapy from the age of eleven until fifty years of age (Nielsen 1999). This may prevent breast development and normalize body hair and beard growth. The testicles are of normal size at birth, but remain around that size all through life. A few males with Klinefelter syndrome have nevertheless produced children. Skeletal maturity in sex chromosome trisomies does not differ significantly from that in a normal population (Webber et al. 1982).

2.3.2.1 Body growth and craniofacial characteristics in 47,XXY males

Birth weight, height and head circumference are smaller in individuals with an extra X chromosome, such as 47,XXY boys, than in normal boys, and head circumference remains below average values to a significant degree (47,XXY -0.7 SD) (Ratcliffe et al. 1994b). Acceleration in height growth is greater than in normal boys up to the age of eight years, owing to accentuated leg length (Nielsen 1999). The pubertal growth spurt in 47,XXY males is similar in magnitude and timing to that in normal boys. They grow taller than normal men, with a mean adult height of 186 cm and 180 cm accordingly, but remain eight to ten centimetres shorter than 47,XYY males (Ratcliffe 1999). In a Finnish study the final height of 47,XXY males was 182 cm (Varrela 1984). The somewhat greater growth acceleration in height is due to relatively increased leg length, and the feminine trunk proportions in 47,XXY males are caused by a decrease in shoulder width (Varrela 1984), and the double dose of X chromosomes inhibits masculine muscular development (Tanner et al. 1959).

The head and face dimensions of 47,XXY males are smaller than those of normal men (Varrela 1984) as is also the cranial base angle (Ingerslev & Kreiborg 1978; Brown et al. 1993). The ramus of the mandible is shorter, but the gonial angle and corpus length are larger than those of normal men, while if compared to normal women there are few contrasts in facial shape apart from mandibular prognathism (Brown et al. 1993). Bimaxillary prognathism and a tendency for a reduction in face height is seen in 47,XXY males. In addition, they have relatively frequently occlusal anomalies, predominantly mesial molar occlusion and anterior open bite (Alvesalo & Laine 1992), and the maxillary and mandibular dental arches are narrow compared to normal men.

34

2.3.2.2 Body growth and craniofacial characteristics in 47,XYY males

Birth weight, height and head circumference of 47,XYY males do not differ from those of normal boys (Ratcliffe 1985). The velocity of height growth increases significantly from two years of age and continues to be high throughout childhood. By the age of seven years the most prominent features are relatively long legs and a small head circumference. The increased height growth is still evident during puberty, which occurs an average six months later than in controls and lasts longer (Ratcliffe et al. 1990; Ratcliffe et al. 1992). Not only height, but virtually all adult body, head and cephalometric dimensions of 47,XYY males are larger than those in normal men with similar body proportions (Varrela & Alvesalo 1985; Grön et al. 1997). 47,XYY males have somewhat narrower shoulders and relatively wider hips and an otherwise generalized size increase (Ratcliffe 1985; Varrela & Alvesalo 1985). The extra Y chromosome in these males affects adult height by about 13 cm compared to normal men, which is close to the difference between normal men and women (Ratcliffe et al. 1992).

47,XYY males have larger craniofacial dimensions than normal men, without any substantial differences in the dimensional ratios or plane angles (Grön et al. 1997). They have a longer cranial base and the cranial base angle is smaller, however. The lower anterior face height is bigger compared to normal men, and the mandible is posteriorly rotated. The foramen magnum is smaller in the sagittal plane than in normal men or women, men having smaller foramen magnum than women. That could be explained by the appositional bone growth in calvaria combined with longer growing bones in men. 47,XYY males have seldom occlusal anomalies, but they tend to have progenia with mesial molar occlusion and negative overjet (Laine et al. 1992), which is accounted for the larger transversal and sagittal maxillary dental arch and mandibular arch length relative to normal men (Laine & Alvesalo 1993). The palatal height and transversal with of the mandibular dental arch are smaller compared to normal men (Laine & Alvesalo 1993).

2.3.3 Female sex reversals (46,XY females)

The testicular feminization syndrome, or females with a male sex chromosome complement, female sex reversals (Therman & Susman 1993) or 46,XY females, can be divided into complete and incomplete forms (Dewhurst 1971). The complete form of androgen insensitivity syndrome involves normal androgen synthesis, the basic endocrine defect being end-organ insensitivity to androgens, which is a genetically determined condition and runs in families. It is a very rare condition and is estimated to occur in one out of 62 400 live-born males (Jagiello & Atwell 1962). These individuals are phenotypically females, and the clinical signs include testes, female external genitalia, puberty with breast development and a blind-ended vagina (Dewhurst 1971). They are females in their psychosexual behaviour, and are usually detected on account of primary amenorrhoea or an inguinal hernia, and occasionally through other affected family members (Dewhurst 1971; Simpson 1976).

35

In the complete form of testicular feminization syndrome in 46,XY females the body dimensions, including stature (mean 171,5 cm) and head circumference, are above the average for normal women (Varrela et al. 1984b). The body proportions are similar with a tendency towards a slimmer figure compared to normal women. Stature is above the expected mid-parental height. The role of the Y chromosome genes in determining stature and head circumference is conceivable in these individuals (Smith et al. 1985; Varrela & Alvesalo 1985).

The growth change in the complete testicular feminization syndrome females is also seen in the dimensions of the cranial base and maxillary complex falling between those of normal men and women, but the length of the mandibular corpus has been measured to exceed even the mean value for normal men and the ramus height resembles that for normal women with an acute gonial angle (Pietilä et al. 1997). The maxillary dental arch is larger in sagittal dimension, and the palate is higher compared to normal women, also the mandibular dental arch is broader (Grön & Alvesalo 1997). The occlusal relationship tend to be mesial with deep bite.

2.4 Tooth growth and an abnormal sex chromosome complement

Studies on families and individuals with abnormal sex chromosome complement and their relatives in the Kvantti research project (Garn & Rohmann 1962; Filipsson et al. 1965; Garn et al. 1964; Alvesalo 1971; Alvesalo et al. 1975a; Alvesalo & Kari 1977; Alvesalo & Portin 1980; Alvesalo & Varrela 1980; Kari et al. 1980; Alvesalo & Tammisalo 1981; Townsend et al. 1984; Alvesalo 1985; Alvesalo et al. 1985; Alvesalo et al. 1987; Mayhall et al. 1991; Mayhall & Alvesalo 1992; Alvesalo 1997; Zilberman et al. 2000), together with molecular research (Lau et al. 1989; Nakahori et al. 1991; Salido et al. 1992), have shown that genes on the human X and Y chromosomes have an effect on the growth of the tooth crown. The Y chromosome promotes formation of both the coronal enamel and dentin, whereas the effect of the X chromosome on crown growth seems to be restricted to enamel (Alvesalo & Tammisalo 1981; Alvesalo 1985; Alvesalo et al. 1985; Alvesalo et al. 1987; Mayhall et al. 1991; Alvesalo 1997; Zilberman et al. 2000). The initiation of dentin formation is preceded by cell proliferations in the inner enamel epithelium, and enamel formation is decisively influenced by cell secretory function (Kraus and Jordan 1965).

Previous studies on females with a lack of sex chromosome material have shown significantly smaller dimensions of the permanent tooth crowns in 45,X, 45,X/46,XX and 46,X,i(Xq) females than in normal women (Varrela et al. 1988; Filipsson et al. 1965; Alvesalo & Tammisalo 1981; Townsend et al. 1984; Mayhall et al. 1991; Midtbø & Halse 1994a). The primary molars also tend to be smaller, at least in 45,X females (Kari et al. 1980). The smaller crown dimension in 45,X, 45,X/46,XX and 46,X,i(Xq) females is mainly due to a thin enamel layer, while the mesio-distal dentin layer is close to that of normal women in 45,X, and 45,X/46,XX females (Alvesalo & Tammisalo 1981; Mayhall et al. 1991; Alvesalo 1985; Zilberman et al. 2000). In 46,X,i(Xq) females the mesio-distal dentin layer is smaller compared to normal women (Mayhall et al. 1991). The change in the labio-lingual dimension is more variable than that in the mesio-distal dimension in

36

these Turner females, and is least affected in 45,X/46,XX females (Varrela et al. 1988; Mayhall et al. 1991; Midtbø & Halse, 1994a). The morphology of the maxillary teeth, especially the incisors, is more variable in Turner women than in controls (Midtbø & Halse 1994a).

Studies on tooth growth in females with the complete form of testicular feminization syndrome (46,XY females) have shown larger crown sizes relative to normal women. Measurements of the thickness of the tooth crown enamel in these females have suggested values close to those in normal men and women, but dentin thickness is similar to that of men and hence far in excess of that in women (Alvesalo 1985), suggesting a Y chromosomal influence on tooth growth apart from androgen action. Alvesalo and de la Chapelle (1981) found that stature and deciduous and permanent tooth crown sizes were reduced in a boy with a deletion in the proximal portion of the long arm of the Y chromosome, compared with another man who also displayed a deletion of the long arm of the Y chromosome, and suggested that the location of the tooth growth-promoting gene is Yq11, which also is the suggested locus for stature.

Studies on deciduous and permanent tooth crown dimensions in 47,XYY boys and men (Alvesalo et al. 1975a; Alvesalo & Kari 1977) have shown bigger tooth crown sizes compared with normal men. Larger crown size in permanent teeth is due to both thicker mesio-distal dentin and enamel layers (Alvesalo et al. 1985). The mesio-distal dimension of upper permanent central incisors, for example, is about five percent larger than in normal men. The bigger crown size of permanent teeth in 47,XXY males (Alvesalo & Portin 1980; Townsend & Alvesalo 1985) is caused by a thicker enamel layer than in normal men or women, while the mesio-distal dentin thickness falls between those of normal men and women (Alvesalo et al. 1991), suggesting an X chromosome gene effect on crown size. The average difference in tooth size between normal men and women is explainable by the postulated differential growth promoting effect of the Y chromosome compared to the X chromosome (Alvesalo 1985). It is of interest, that the effect of the sound X chromosome on metric enamel formation shows similar class of magnitude to that of the Y chromosome, whereas, in crown dentin formation the influence of the Y chromosome is clearly more than that of the X chromosome (Alvesalo 1997).

The crown morphology of 47,XYY males is changed in that the degree of shovelling of the maxillary permanent lateral incisors is bigger and the palatal fossa is deeper than in their relatives, while the lateral incisors of 45,X females are less shovel-shaped than in normal women and the central incisors have a shallower fossa in addition to the tendency towards less cusps in the molars (Kirveskari & Alvesalo 1981) and simplified tooth crown shape (Kirveskari & Alvesalo 1981; Midtbø & Halse 1994a). Midtbø and Halse (1994a) found an altered mamelon pattern in Turner women, especially in the incisal edge of the maxillary lateral incisors, together with atypical mesiobuccal cusps and nippled cusp tips of the maxillary canines and premolars, whereas the Carabelli trait in the maxillary first molars was found far less often than in a normal Finnish population (Alvesalo et al. 1975b; Nakayama et al. 2005). Sex chromosomes have an effect mainly on the cusp basal area rather than cusp height. The cusp basal area is smallest in 45,X females, with the sharpest cusps, becomes larger in normal women and men, and is even larger in 47,XYY males, who have the bluntest cusp form (Mayhall & Alvesalo 1995).

Permanent tooth root lengths are shorter in 45,X, 45,X/46,XX and 46,X,i(Xq) females (Filipsson et al.1965; Midtbø & Halse 1994b) relative to normal women, while the

37

premolars (Varrela 1990; Midtbø & Halse 1994b) and lower first molars (Midtbø & Halse 1994b) of 45,X females show more root components in separate forms and higher frequency than in normal women, and also molariform mandibular premolars with mesial and distal roots. Taurodontism appears to occur in 45,X (Varrela et al. 1990), 45,X/46,XX females and 47,XYY males (Alvesalo & Varrela 1991) as in a normal population with a frequency of 0.5-5 percent (Jaspers & Witkop 1980). Exceptionally, the pulp chamber of the mandibular second premolar is often longer in 45,X females and 45,X/46,XX females compared to normal women (Midtbø & Halse 1994b; Varrela 1992). The prevalence of taurodontism increases with additional X chromosomes (Jaspers & Witkop 1980; Varrela & Alvesalo 1989), and hypotaurodontic teeth appear to occur in about one third of mainly second molars in 47,XXY males (Varrela & Alvesalo 1988).

3 Aim of this research

The general aim of the research was to study tooth root growth in individuals with an abnormal sex chromosome complement and in 46,XY females who are insensitive to androgens. The approach may also provide some clues for a further insight into the development of sexual dimorphism in human growth. The underlying hypothesis was that the effect of the X and Y chromosomes on tooth crown growth is also expressed in root growth.

More specifically, the objectives were:

− to determine completed permanent tooth root lengths in 45,X/46,XX females, 47,XYY and 47,XXY males, 46,XY females and normal men and women from panoramic radiographs

− to deduce the timing of the influence of an abnormal sex chromosome complement and of androgen insensitivity in 46,XY females on tooth root growth

− to deduce the relative magnitude of the influence of the X and Y chromosome genes on root growth

4 Subjects and Methods

4.1 Population

The subjects were participants in professor Lassi Alvesalo’s research project, Kvantti (Kromosomaalisen VAikutukseN Tutkimus-projekTI) on individuals with abnormal sex chromosome constitution and 46,XY females (here called individuals) and their first degree relatives (here called relatives). The individuals and relatives were from different parts of Finland. Cytogenetic diagnosis of the individuals had been carried out for for medical reasons. The Institutional Review Board of the Medical Faculty, University of Turku, Finland, had reviewed and approved the protocol, of which the individuals and their relatives were informed. The examinations were carried out during a time period beginning 1974 and continuing up to 1987 with the individuals’ and relatives’ consent, and they were not at risk in any way.

The individuals, who were 47,XYY males, 46,XY females, 47,XXY males and 45,X/46,XX females, are presented here by groups as in the original papers, referred to by the Roman numerals (I-IV). For comparisons the relatives were chosen on a paired basis, one relative of the same phenotypic sex for each individual and comparisons were also extended to opposite sex in the group of 47,XXY males with extra X or Y chromosome. In the selection of relatives the preference was put on the same generation (sister or brother) prior to the parents and in the case of several siblings of the same phenotypic sex the criteria was to choose the one primarily with full dentition and minimal restorations and good quality of the radiograph. The population controls (men and women) comprised relatives other than the group of individuals in question. (Table 2)

According to anamnestic information ten out of the fifteen 45,X/46,XX females had received hormone therapy (mainly oestrogen), and four out of the eight 46,XY females had received oestrogen therapy during or after puberty. 46,XY females have a pubertal growth spurt typical of normal women both in magnitude and timing (Frank 2003). Oestrogens are responsible for the normal development of the female secondary sex characteristics and act on the fertilization process during the menstrual cycle.

Tabl

e 2.

The

indi

vidu

als

acco

rdin

g to

thei

r cy

toge

netic

dia

gnos

es, t

heir

rela

tives

and

pop

ulat

ion

cont

rols

. The

Rom

an n

umer

als

indi

cate

th

e or

igin

al a

rtic

les.

Indi

vidu

als

Rel

ativ

es

Con

trol m

en

Con

trol w

omen

St

udy

grou

ps

N

Age

m

ean

SD

Ran

ge

N

Age

m

ean

SD

Ran

ge

N

Age

m

ean

SD

Ran

ge

N

Age

m

ean

SD

Ran

ge

47,X

YY

mal

es

(I)

15

18.6

7.

38

10.0

-36.

7 9a

27.9

12

.64

11.9

-42.

7 22

23

.7

12.4

1 11

.6-4

5.6

26

26.4

10

.60

9.7-

55.6

47,X

XY

mal

es

(IV

) 49

30

.7

11.7

1 10

.2-5

7.6

22b

8c 41

.0

39.7

15

.53

5.64

13

.5-6

7.5

31.2

-50.

6 35

25

.5

12.4

1 11

.6-4

5.6

46

27.6

10

.66

9.7-

55.6

46,X

Y fe

mal

es

(II)

8

25.1

9.

57

9.3-

41.7

5d

24.8

10

.60

16.9

-43.

5 56

30

.9

15.7

5 11

.6-6

7.5

50

29.3

11

.14

9.7-

44.1

45,X

/46,

XX

fe

mal

es (I

II)

15

23.4

8.

42

7.1-

41.0

10

e 24

.5

7.55

15

.1-3

7.4

- -

- -

47

29.8

11

.37

9.7-

59.1

N =

num

ber i

n st

udy

grou

ps, M

ean

age

in y

ears

, SD

= st

anda

rd d

evia

tion,

Ran

ge =

min

imum

- m

axim

um (y

ears

), a

= 4

fath

ers a

nd 5

bro

ther

s, b

= 16

fath

ers a

nd 6

bro

ther

s, c

= 4

mot

hers

and

4 si

ster

s, d=

5 si

ster

s, e=

2 m

othe

rs a

nd 8

sist

ers

40

41

4.2 Methods

4.2.1 Dental panoramic tomogram

A dental panoramic tomogram can provide information on the whole dentition in one radiograph. The parabolic basic form of the image layer in orthopantomography is obtained by means of a rotary movement taking place on three axes in succession. The form of the image layer can be changed to some extent, especially by means of the profile guide of the orthopantomograph and by regulating the speed of the film (Tammisalo 1964b). It was proposed empirically by Tammisalo & Nieminen (1964) that the position of the centre of the sharply depicted object plane is dependent on the ratio between the velocity of the film in relation to the beam and the velocity of the beam in relation to the object. This empirical postulate, the outcome of a mathematical analysis by Tammisalo (1964a) of the position and form of the image layer in images exposed with one specific device, the Orthopantomograph 2, was later confirmed in experimental tests (Sjöblom & Welander 1978; Welander & Wickman 1978). Distortion tolerance is better when the projection radius is large, as is the case with the lateral parts of an image (Welander & Wickman 1978), but the errors appearing in the reproduction of the inner surface of the image layer on a horizontal plane are greater than those of the outer surface, because the displacement and the difference in speed between the film and projection have an effect similar to a change in dimensions. On the vertical plane, dimensional reproduction of the object layer is dependent on the projection alone, any enlargement of the points situated in the basic plane being linear, and the magnification is 31.7 percent (Tammisalo 1964a). Distortion of the image in rotational panoramic radiography with respect to the vertical dimension has also been analysed by Tronje et al. (1981) and Stramotas et al. (2002), who stress the importance of the proper position of the patient in the device during exposure.

Larheim et al. (1984), investigating the reproducibility of radiographs with the Orthopantomograph 5, found the variability of measurements assessed from repeated radiographs to range from 0.65 to 0.85 millimetres, or 2.4 to 3.1 percent of the mean radiographic tooth length in different patient groups. The measurement error ranged from 0.43 to 0.56 millimetres, indicating that the main source of error inherent in the method was recognition of the reference points. Small differences were found between the tooth groups and between the right and left sides. Thanyakarn et al. (1992) observed that radiographic tooth length measurements are highly influenced by observer performance, the variation being higher for measurements of the maxillary teeth (0.3-1.9 mm) than for the mandibular teeth (0.3-0.9 mm) in the case of most observers. The highest variation was for the palatal root of the maxillary first molar.

The panoramic dental radiographs had been taken of all the patients and their relatives in this study by one person at the Institute of Dentistry, University of Turku, within a period of approximately ten years starting in 1974, following a standardized procedure and with the same machine, an Orthopantomograph 3, Palomex. The magnification ranged between 1.28-1.31 (28-31 percent) over the whole image layer of the panoramic radiograph.

42

4.2.2 Root length measurement

The root lengths of the teeth of the individuals and controls were measured from the dental panoramic radiographs, and crown heights were measured at the same time for further studies (Fig. 4). The aim was to measure all the teeth with complete root formation on both sides of the jaws except for the third molars. A computer-aided digital system was first undertaken to measure the root lengths on the tomographic radiographs. A videocamera was used to copy the radiograph on a light table and the root length was calibrated. However, that method was found to be inadequate for defining the outlines of the teeth on these radiographs because of weak contrast in places.

In the present study the following method was used: the radiograph was placed on a light table and observed in a dark room, and magnifying lens (2x) was used to determine the outlines of the teeth on the radiograph. The outlines were marked with a special, sharp pencil for use with plaster (Schwan All Stabilo 8008). Measurements were all made in the same manner with a sliding digital calliper (Mitutoyo, digimatic 500-123U, CD-15B, England) to an accuracy of 0.01 mm. The measured value was rounded to one decimal in the results in the following way: if the second decimal value was from one to four the first decimal remained unchanged and if it was five or more the first decimal was raised. The measurements of root lengths were made on a perpendicular between two parallel lines, a line touching the outermost part of the root and a line joining the mesial and distal cervical margins of the enamel. The value of the measurement was taken according to the outer edges of the traces of the pencil on the radiograph. Root length refers to the longest root on the radiograph in the case of premolars and the longest mesial root in the case of molars. All the drawings and measurements were made by the present author (RL). (Fig. 5).

The cervical margin was determined in the manner as Lind (1972) has described: “points of intersection between outer contours of root and crown connected by a straight line” combined with scrutinizing the lighter shade of enamel compared to dentin in a radiograph. The root lengths and crown heights were measured as the projections of the cusp tip and root apex to the film. The projections were considered the maxillary palatal cusp tip and mandibular distal cusp tip in molars and the longer cusp tip in premolars. All separate roots were first measured except the palatal root of the maxillary first molar because of its great enlargement in this kind of radiograph. In the case of two roots on the top of the other on the radiograph the longest dimension was chosen to be measured. Only mesial root length was measured in the case of multi-rooted teeth with mesial and distal root. (Fig. 5b)

Teeth that were partly outside the image layer in the panoramic radiograph or showed obvious distortion because of being on its inner or outer surface (Tammisalo 1964b), and teeth with incomplete root formation, were excluded. The dilacerated roots were measured as perpendicular lengths as described above. A couple of impacted canine teeth

43

Fig. 4. A dental panoramic tomogram of a Klinefelter boy (47,XXY) aged 16 years. The outlines of the teeth were marked using a special pencil for plaster, as also was a line joining the mesial and distal cervical margins of the enamel.

a.

Fig. 5. Root lengths were measured by reference to lines marked on the tomographic radiographs (a) as described in the text and shown in the picture (b).

with a closed apex and good posture in the radiograph were measured (Haavikko 1970). Permanent tooth root lengths may be affected by several external factors, which could bias the results. Orthodontic treatment, especially with fixed appliances, may cause root resorption, as also can traumatic occlusion, bruxism, nail-biting, trauma, apical infection

44

or root treatment, for instance. Teeth with apparent root resorption occurring for any reason were not included.

Acellular cementum is formed on the root surface until the tooth reaches the occlusion, at which point the proliferation of the epithelial root sheath is reduced and it may become entrapped within the forming matrix of cellular cementum (Thomas 1995). Cellular cementum formation continues after the root form is complete. The line between apical cementum and dentin was evaluated while marking the outlines of the roots and the apical cementum layer was excluded from the present root length determinations.

4.2.3 Reliability of the measurements

The reliability of the measurements was examined by performing double determinations on a total of 45 dental radiographs from the ”Kvantti” research material, representing adult 45,X females and their female and male relatives, with fifteen persons in each group. The measurements were made without knowing the patient group by the present author at an interval of two weeks, the marked line joining the mesial and distal cervical margins of the enamel on each tooth being rubbed out after the first measurement and determined again and re-drawn for the second measurement. The drawing of the outline of crown and root remained untouched.

The standard deviation of the differences between the repeated measurements was calculated and 95% of the differences were expected to lie within the limits of ± 2 SD. Scatterplot graphics were used to determine the limits of agreement (Bland and Altman 1986).There were only two out of the total of 28 teeth in maxillary anterior region that showed more variation than ± 2 SD, the standard deviation remaining mainly within the limits of ± 3 SD, with only one point in the intervals ± 3 - 4 SD. According to these calculations the root length measurements lied within the assumed limits.

The absolute error values for the root length measurements ranged from 0.35 mm to 0.75 mm, the corresponding percentages being 1.95 and 5.11. The values were considered acceptable for further measurements.

The reproducibility of the double determinations of root length was expressed in terms of the method error statistic (Dahlberg 1948). The difference between the first and second measurements was also analyzed with coefficients of intra-class correlation (Table 3).

45

SDd = standard deviation d1 = original measurement value d2 = repeated measurement value

N = number of patients

Table 3. Coefficients of intra-class correlation between the first and second measurements of tooth root lengths.

Tooth Maxilla Mandible Right second molar 0.948 0.912 first molar 0.889 0.933 second premolar 0.954 0.957 first premolar 0.927 0.890 canine 0.958 0.949 lateral incisor 0.928 0.945 central incisor 0.977 0.948 Left central incisor 0.975 0.942 lateral incisor 0.890 0.955 canine 0.981 0.970 first premolar 0.894 0.965 second premolar 0.950 0.973 first molar 0.932 0.938 second molar 0.966 0.884

4.2.4 Statistical analyses

The SPSS package 12.0 (Statistical Package for the Social Sciences, CA, USA) was used for the statistical analysis. Mean values for root length were calculated and compared between the individuals, relatives and population controls using the two-tailed T-test for equality of means. Results were considered statistically significant when p was less than 0.05. The analysis of variance (ANOVA) was used to find out the significance of differences between the individual and population control groups. Duncan’s multiple comparison procedure was used to itemize the differences between the study groups as homogenous subgroups were concerned with p equal to 0.05.

( )21 2

2d

d dSD

N

⎛ ⎞−⎜ ⎟=⎜ ⎟⎝ ⎠∑

5 Results

The results are presented by study groups in the original papers and referred to here by the Roman numerals indicating these papers (I-IV). The summary tables (Tables 4 and 5) present the means, numbers and standard deviations for the tooth root length measurements performed on the individuals with sex chromosome abnormalities and the population controls (men and women). In these summary tables the population controls comprise parents and siblings of all the individuals grouped together and are taken to represent the normal population. In the original articles the population controls comprise relatives of the other individuals. The individuals and their paired relatives of the same sex were compared in each paper to approach the problem of normal variation.

The analysis of ANOVA indicated that the differences in tooth root length between the individual and control groups were highly significant. Two-tailed t-test showed differences between separate groups of individuals and control groups, and these differences were further supported by the test of Duncan.

5.1 Root lengths in the permanent teeth of 47,XYY males (I)

The mean permanent tooth root lengths in 47,XYY males were larger than those for the population control men (Tables 4 and 5). The differences were significant in eleven out of the total of 28 comparisons, and the actual relative metric differences in the mandible exceeded those in the maxilla in most instances. Visual inspection of the root morphology on the panoramic radiographs of the 47,XYY males did not reveal any major deviations from normality.

Comparisons of the mean root lengths between nine 47,XYY males and their brothers or fathers showed the former measurements to be larger, with only one exception. The differences were significant in half of the comparisons, and the values for the relatives came closer to those for the population controls than to those for the 47,XYY males (Table 2 in paper I).

The root lengths of the population control men were in most cases larger than those of the control women (Tables 4 and 5), the differences being significant in 15 out of the total of 28 comparisons. The excess root growth in 47,XYY males relative to normal men was

47

in most cases greater than the difference between the normal men and women, except in the upper incisors. The mean percentage difference in all root lengths between sexes was four percent, and that between the 47,XYY males and normal men nine percent.

5.2 Root lengths in the permanent teeth of 46,XY females (II)

The mean permanent tooth root lengths in 46,XY females were larger than those for the population control women (Tables 4 and 5), the difference being significant in eleven out of the total of 28 comparisons, with values similar to those in the population control men. The 46,XY females had significantly longer tooth roots than their sisters in nine out of the total of 28 comparisons (Table 2 in paper II). Visual inspection of the root morphology on the panoramic radiographs for the 46,XY females did not reveal any major deviations from normality.

5.3 Root lengths in the permanent teeth of 45,X/46,XX females (III)

The mean root lengths of the permanent teeth in the 45,X/46,XX females were in most cases less than those of the population control women (Tables 4 and 5), the differences being significant in 23 out of the total of 28 comparisons, and the relative differences in the maxilla exceeded those in the mandible in most instances. The mean percentage difference for all root lengths between the 45,X/46,XX females and the population control women was ten percent. The 45,X/46,XX females had shorter tooth roots than their sisters or mothers, with only one exception (Table 2 in paper III). The differences were significant in half of the comparisons, and the values for the relative women were closer to those of the population controls than to those for the 45,X/46,XX females.

5.4 Root lengths in the permanent teeth of 47,XXY males (IV)

The mean permanent tooth root lengths in the 47,XXY males were larger than in the population control men (Tables 4 and 5), the difference being significant in 12 out of 14 comparisons in the mandible and in the maxillary premolars and molars, and the absolute values for the maxillary incisors were higher than those for the control men. The 47,XXY males also had significantly longer tooth roots than the population control women in all the measurements. The mean difference in all root lengths between the 47,XXY males and the population control men and women was eight and eleven percent, respectively.

The 47,XXY males had longer tooth roots that their relative men in 22 out of the total of 28 measurements (Table 2 in paper IV), the absolute lengths of the tooth roots in the relative men falling mainly between those of the population control men and the 47,XXY males. Visual inspection of the root morphology on the radiographs of the 47,XXY males did not reveal any major deviations from normality, but a minor deviation in the form of taurodontic teeth was observed in 30 percent of cases.

Tabl

e 4.

Sum

mar

y ta

ble

of p

erm

anen

t too

th r

oot l

engt

h (a

) m

easu

rem

ents

of m

axill

ary

teet

h fo

r th

e in

divi

dual

s w

ith s

ex c

hrom

osom

e ab

norm

aliti

es, 4

6,XY

fem

ales

, and

nor

mal

men

and

wom

en (I

-IV)

.

47,X

YY♂

46

,XY♀

47

,XX

Y♂

46

,XY♂

46

,XX♀

45

,X/4

6,XX♀

To

oth

root

M

axill

a M

ean

N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Rig

ht se

cond

mol

ar

16.4

9

/ 2.0

16

.4

3 / 2

.3

17.1

24

/ 2.

2 15

.6

43 /

2.1

14.8

36

/ 1.

9 13

.7

13 /

1.6

first

mol

ar

16.4

12

/ 2.

5 14

.9

3 / 1

.6

17.4

23

/ 1.

8 15

.2

41 /

1.8

14

.7

35 /

1.9

13.0

14

/ 1.

3

seco

nd p

rem

olar

20

.2

11 /

3.0

19.9

2

/ 1.8

19

.5

18 /

2.6

17.3

41

/ 2.

4 17

.2

34 /

1.7

14.8

14

/ 2.

3

first

pre

mol

ar

18.6

12

/ 2.

2 18

.0

4 / 1

.9

19.9

29

/ 2.

2 18

.7

35 /

2.3

17.3

32

/ 1.

8 15

.5

13 /

1.7

cani

ne

24.6

14

/ 2.

8 23

.9

4 / 4

.1

23.3

30

/ 2.

9 24

.0

42 /

2.2

22.0

40

/ 2.

1 19

.5

14 /

2.5

late

ral i

ncis

or

19.2

11

/ 1.

9 19

.8

4 / 0

.9

19.8

28

/ 1.

9 19

.6

39 /

2.2

18.3

36

/ 1.

5 16

.0

14 /

2.5

cent

ral i

ncis

or

20

.7

15 /

2.0

21.7

4

/ 2.3

21

.0

33 /

2.4

20.3

47

/ 2.

3 18

.9

39 /

1.4

15.9

15

/ 3.

2

Left

cent

ral i

ncis

or

20.9

15

/ 1.

9 21

.1

5 / 2

.4

21.1

32

/ 2.

4 20

.3

44 /

2.2

19.1

38

/ 1.

2 16

.3

15 /

2.9

late

ral i

ncis

or

20.2

13

/ 1.

8 19

.0

3 / 1

.5

20.0

28

/ 2.

0 19

.5

45 /

1.9

18.4

35

/ 1.

7 16

.4

15 /

2.4

cani

ne

25.4

10

/ 1.

2 22

.7

4 / 2

.7

23.4

25

/ 2.

7 24

.1

45 /

1.9

22.2

39

/ 1.

9 19

.7

14 /

1.6

first

pre

mol

ar

18.1

11

/ 3.

7 19

.1

4 / 4

.5

19.9

24

/ 1.

9 18

.6

37 /

2.3

17.0

33

/ 1.

8 15

.0

11 /

2.4

seco

nd p

rem

olar

18

.9

12 /

2.7

19.5

3

/ 4.3

19

.7

24 /

2.4

17.4

40

/ 2.

5 17

.2

35 /

1.7

14.7

12

/ 2.

2

first

mol

ar

16.0

13

/ 2.

0 14

.7

4 / 3

.9

16.8

21

/ 1.

7 15

.0

39 /

1.9

14.5

34

/ 1.

8 13

.1

13 /

1.1

seco

nd m

olar

15

.6

10 /

1.3

15.1

3

/ 3.0

17

.4

27 /

2.4

15.0

40

/ 2.

5 14

.3

34 /

1.9

13.5

14

/ 1.

4

Mea

n=m

ean

root

leng

th (m

m),

N=n

umbe

r of i

ndiv

idua

ls w

ith se

x ch

rom

osom

e ab

norm

aliti

es a

nd 4

6,X

Y fe

mal

es o

r nor

mal

con

trols

, SD

=sta

ndar

d de

viat

ion

a) R

oot l

engt

h re

fers

to th

e lo

nges

t roo

t in

the

case

of t

wo

root

s on

the

top

of th

e ot

her a

nd th

e m

esia

l roo

t in

the

case

of m

ulti-

root

ed te

eth

on th

e ra

diog

raph

. 47

,XY

Y♂

=a m

ale

with

an

extra

Y c

hrom

osom

e 46

,XY♀

=a fe

mal

e w

ith a

mal

e se

x ch

rom

osom

e co

mpl

emen

t and

com

plet

e fo

rm o

f and

roge

n in

sens

itivi

ty sy

ndro

me

(a fe

mal

e se

x re

vers

al)

47,X

XY♂

=an

indi

vidu

al w

ith a

n ex

tra X

or Y

chr

omos

ome

(Klin

efel

ter m

en)

46,X

Y♂

= po

pula

tion

cont

rol m

en, r

elat

ives

of a

ll in

divi

dual

s gro

uped

toge

ther

; 46,

XX♀

=pop

ulat

ion

cont

rol w

omen

, rel

ativ

es o

f all

indi

vidu

als g

roup

ed to

geth

er

45,X

/46,

XX♀

=a m

osai

c fe

mal

e in

whi

ch a

seco

nd c

ell l

ine

has t

he c

hrom

osom

e co

nstit

utio

n 45

,X (T

urne

r wom

en)

48

Tabl

e 5.

Sum

mar

y ta

ble

of p

erm

anen

t too

th r

oot l

engt

h (a

) mea

sure

men

ts o

f man

dibu

lar

teet

h fo

r th

e in

divi

dual

s w

ith s

ex c

hrom

osom

e ab

norm

aliti

es, 4

6,XY

fem

ales

, and

nor

mal

men

and

wom

en (I

-IV)

.

47,X

YY♂

46

,XY♀

47

,XX

Y♂

46

,XY♂

46

,XX♀

45

,X/4

6,XX♀

To

oth

root

M

andi

ble

Mea

n N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Mea

n N

/ SD

Rig

ht se

cond

mol

ar

19.5

9

/ 2.8

19

.5

4 /

0.9

18.5

22

/ 2.

0 17

.8

33 /

2.2

17.2

33

/ 1.

4 16

.0

13 /

2.0

first

mol

ar

20.2

11

/ 2.

2 18

.3

4 / 2

.0

19.7

24

/ 1.

7 18

.9

35 /

2.1

17.7

28

/ 1.

3 16

.4

9 /

2.5

seco

nd p

rem

olar

20

.9

5 / 1

.9

20.8

4

/ 1.2

20

.7

22 /

2.3

19.3

44

/ 2.

6 18

.2

36 /

1.9

16.5

11

/ 2.

9

first

pre

mol

ar

19.8

13

/ 1.

8 19

.1

6 / 0

.9

19.9

39

/ 2.

0 18

.9

52 /

2.5

17.6

41

/ 1.

8 16

.2

14 /

1.3

cani

ne

23.8

12

/ 2.

5 21

.7

7 / 1

.8

22.6

39

/ 2.

2 22

.5

54 /

2.3

19.8

42

/ 2.

0 17

.6

13 /

1.6

late

ral i

ncis

or

18.8

12

/ 1.

9 16

.7

8 / 1

.2

18.6

41

/ 1.

9 17

.6

56 /

2.0

15.7

45

/ 1.

7 14

.0

14 /

1.1

cent

ral i

ncis

or

17

.8

13 /

1.8

16.1

6

/ 0.8

17

.6

41 /

1.8

16.1

52

/ 2.

1 14

.5

46 /

1.7

13.5

13

/ 1.

7

Left

cent

ral i

ncis

or

16.8

12

/ 2.

4 17

.0

8 / 1

.7

17.9

41

/ 1.

9 15

.6

51 /

2.1

14.5

45

/ 1.

7 13

.8

14 /

1.7

late

ral i

ncis

or

18.9

14

/ 2.

1 17

.4

7 / 1

.3

18.7

43

/ 2.

0 17

.6

53 /

2.3

15.7

44

/ 1.

8 14

.2

14 /

1.4

cani

ne

24.0

13

/ 2.

4 22

.6

7 / 1

.8

22.5

42

/ 2.

7 21

.7

54 /

2.8

19.5

40

/ 1.

7 17

.7

14 /

2.2

first

pre

mol

ar

20.0

10

/ 1.

5 19

.0

6 / 1

.6

19.4

37

/ 1.

9 18

.9

54 /

2.5

17.9

42

/ 1.

8 16

.6

13 /

2.0

seco

nd p

rem

olar

21

.7

9 / 2

.7

19.8

4

/ 2.8

21

.1

20 /

2.1

19.3

45

/ 2.

8 18

.8

39 /

1.7

17.2

13

/ 2.

0

first

mol

ar

20.3

10

/ 1.

8 19

.2

3 / 3

.2

20.3

19

/ 1.

6 18

.9

35 /

1.7

17.8

29

/ 1.

5 16

.2

12 /

1.3

seco

nd m

olar

18

.5

6 / 1

.9

19.2

3

/ 0.8

18

.9

22 /

2.0

17.2

32

/ 2.

3 17

.2

30 /

1.5

15.6

13

/ 1.

8

Mea

n=m

ean

root

leng

th (m

m),

N=n

umbe

r of i

ndiv

idua

ls w

ith se

x ch

rom

osom

e ab

norm

aliti

es a

nd 4

6,X

Y fe

mal

es o

r nor

mal

con

trols

, SD

=sta

ndar

d de

viat

ion

a) R

oot l

engt

h re

fers

to th

e lo

nges

t roo

t in

the

case

of t

wo

root

s on

the

top

of th

e ot

her a

nd th

e m

esia

l roo

t in

the

case

of m

ulti-

root

ed te

eth

on th

e ra

diog

raph

. To

spec

ify th

e di

ffer

ent s

tudy

gro

ups s

ee T

able

5.

49

6 Discussion

In addition to the population controls, the present study groups included the first-degree relatives of the individuals with sex chromosome abnormalities and 46,XY females as controls. This allowed consideration of the possibility that the mean root lengths in relatives may deviate from the mean values for the population. One relative of the same phenotypic sex for each individual was chosen and the comparisons were also extended to the opposite sex if possible. The other criteria were age, generation and the number of teeth available. The relatives’ values were in most cases closer to the values for the population controls than to those for the individuals with extra sex chromosomes, such as 47,XYY and 47,XXY males and 45,X/46,XX females with a shortage of sex chromosome material. The 46,XY individuals who are insensitive to androgens and phenotypically females had in most cases significantly longer tooth roots than their female relatives. All in all, the root lengths of the relatives fell within the normal range of variation.

The beam in a dental panoramic radiograph is not perpendicular to the labiolingual direction of the tooth in most instances, and the inclination of the tooth varies between dentitions and individuals. Lind (1972), having studied the effect of varying inclination and slight to moderate rotation of the tooth in relation to the film or beam, found that these factors did not influence the root/crown ratio by more than 0.1. An even distribution in these variables was assumed between the study groups. The magnitude of the tooth inclination in relation to the beam and film can be considered here to lie between the limits suggested by Tronje et al. (1981): -20 and 30 degrees in the mandible and -15 and 45 degrees in the maxilla.

The fact that the mean root lengths of antimeric teeth differed to some extent may be due to the sample sizes, the varying numbers of measurements available and general technical reasons. Certainly, the measurements of natural tooth roots also show differences between the mean lengths for antimeric teeth (Selmer-Olsen 1949). Permanent tooth root lengths may be affected by several external factors, which could bias the results. According to anamnestic information (gathered during the years 1974-1987), the individuals or their relatives had not had any orthodontic treatment, at least not with fixed appliances, before the examination procedures. Similarly, anamnestic information on the population controls suggested that they had not undergone orthodontic therapy. This is supported by the fact that at the time in question there were only very few

51

dental offices in Finland where fixed appliance orthodontics, or orthodontics in general, were carried out. Regarding the possible effects of other external factors, the assumption was made of an even distribution between the study groups.

The main source of error in tooth length measurements in repeated dental panoramic radiographs has been found to lie in the recognition of the reference points. Larheim et al. (1984) found only small differences between the tooth groups and between the right and left sides in tooth lengths in repeated dental panoramic exposures, but they felt that it was necessary to mark some reference points with a pencil because of difficulties in recognition. The vertical plane in an orthopantomograph was considered trustworthy under standardized circumstances (Larheim et al. 1984; Tronje et al. 1981). The problem in finding the reference points was handled here by marking the outlines of the teeth directly on the radiograph, with special emphasis on the apical dentin/cementum border, where there is a slight difference in radiodensity. Furthermore, all the radiographs were taken using the same machine and by same person, which is important for obtaining comparable radiographs with this method, where the positioning of the patient in the machine is most important. Measurement of the tooth length on a panoramic dental tomogram has been considered a suitable method and permitted us to compare root lengths between the study groups.

The accuracy of marking the cervical line on a dental panoramic radiograph to measure the root length was evaluated by performing the measurements twice and rubbing the cervical line out after the first measurement and re-drawing it for the second. The coefficients of intra-class correlation of the root lengths between the first and second measurements were considered reliable for further measurements.

The root lengths were measured as projections of the root apex to the film. Only the mesial root was included in the calculations in the case of multi-rooted teeth with mesial and distal roots. In the case of two roots on the top of each other on the radiograph, the longest dimension was chosen for measurement, as it is impossible to define the labio-lingual order in this kind of panoramic radiograph. Mineralization of the hard tissues during tooth development starts in the mesial part of the crown and it is evident that the same order continues during root development, expressing a more invariable part of the tooth.

All in all, the results showed longer final permanent tooth roots in the 47,XYY and 47,XXY males, while the roots in the 45,X/46,XX females were shorter than in normal men and women, respectively. The root lengths of the 46,XY females were longer than in normal women and similar to those for normal men. The differences in all measured tooth root lengths between the individual and control groups were very clear according to the analysis of ANOVA. When the Duncan test was used to compare the individuals with sex chromosome abnormalities and population controls, the root lengths of the canines, maxillary central incisors, and mandibular lateral incisors clearly differed between the normal men, women and 45,X/46,XX females with a shortage of sex chromosome material, the men having the longest roots, the mosaic females the shortest and the normal women lying between them. The root lengths in all the teeth measured clearly differed between the mosaics (45,X/46,XX females) and the trisomies (47,XXY and 47,XYY males).

The root lengths in the population control men were longer than those in the population control women, as observed previously (Selmer-Olsen, 1949). Extremely

52

short roots are found more frequently in girls and extremely long roots in boys. The mean difference between the sexes in the present root lengths was five percent, which is parallel with the six percent reported by Garn et al. (1978) in mandibular canines, premolars and molars. The present results regarding tooth root lengths in individuals with extra and shortage of sex chromosome material and population controls strongly indicate that the promoting effect of the Y chromosome on growth in root length is greater than that of the X chromosome. This differential effect may lead to the expression of sexual dimorphism in root size.

It is known that the crown sizes of the canines show the greatest difference between the sexes. The root length difference between the present normal men and women in the canines was found to be twice as great as the mean root length difference between these groups, being significant according to the test of Duncan. The roots of the canines were longer in the 47,XYY males, with an extra Y chromosome, than in the normal men, while in the 47,XXY males and 46,XY individuals (insensitive to androgens and phenotypically females), the mean root lengths of the canines were close to those in the normal men. On the other hand, the roots of the canines in 45,X/46,XX females, with a shortage of sex chromosome material, were very much shorter than in the normal women, which is parallel with earlier reports based on this and other Turner female groups (Midtbø & Halse, 1994b).

The root morphology did not reveal any major deviations from normality in either of the study groups. The 45,X/46,XX females showed the same tendency in root morphology as described in the 45,X females. A mild form of taurodontism was found in one third of the roots of the second molars of the 47,XXY males, which is parallel with the earlier findings that the incidence of taurodontism increases in the presence of an extra X chromosome. The plump shape of the roots, especially in the maxillary central incisors, found in a normal population with short roots (Lind 1972) does not appear in the form of reduced root length in the case of Turner females.

Earlier results regarding tooth crown sizes in 47,XXY males had indicated smaller mesio-distal crown dentin thickness than in normal men, which was considered to result from the “retarding” effect of an extra X chromosome (Alvesalo et al. 1991). However, root dentin formation in the 47,XXY males was greater than that in normal men or women. Only the root lengths of the canines in the present normal men exceeded the values for the 47,XXY males. On the other hand, the enamel layer in the tooth crowns of the 47,XXY males was markedly thicker than in the normal men or women, as is also the case in 47,XXX females. The mesio-distal crown dentin size in the canines of 47,XXX females has been shown to be slightly decreased, too (Alvesalo et al. 1987). These findings need further discussion. Patients with dentinogenesis imperfecta type I with normal enamel formation but defects in the formation of dentin appear to have smaller tooth crowns and surely show shorter than normal roots (J. Waltimo-Sirén, personal communication), which is suggesting of a mesenchymal participation in the crown growth.

Turner females, including mosaics, 45,X/46,XX females, have smaller tooth crown sizes with simplified tooth crown shapes and a tendency towards less ordinary cusps in the molars and shorter roots, due to shortage of sex chromosome material by comparison with normal women. On the other hand, they have extra cusps and nippled cusp tips in the premolars and a mamelon pattern of the incisal edges of the lateral incisors and

53

molariform mandibular premolars, and the incidence of root components in dissimilar variants is increased. It may be that this diversity in the morphology of the tooth crown and root associated with a shortage of sex chromosome material is affected by different genetic information than the small and reduced crown size and short roots.

A relationship between tooth size and body size has been suggested (Garn et al.1968). There is a clear size difference in stature, tooth crown dentin mesio-distal diameter and root length between normal men and women, with normal men showing larger values. In individuals with a shortage of sex chromosome material, stature, the enamel layer in the tooth crowns and tooth root lengths are all smaller than in normal women. On the other hand, an extra X chromosome increases stature, with modified body proportions, the enamel becomes thicker and the tooth roots are longer. An extra Y chromosome has the greatest influence on stature, resulting in similar body proportions as in normal men, bigger tooth crown sizes and longer roots.

Earlier dental studies have shown that the Y chromosome genes promote growth of the permanent tooth crown, both the enamel and the dentin, whereas the effect of the X chromosome genes on crown formation seems to be restricted mainly to the enamel. The formation of enamel is decisively influenced by the cell secretory function, and dentin formation in the crown by cell proliferations or mitotic activity. It has been suggested that the effect of the Y chromosome, particularly in increasing cell proliferations, explains the expression of sexual dimorphism in crown size and shape, maturation and the number of teeth, i.e. supernumerary permanent teeth are approximately twice as common in normal males as in normal females and permanent ordinary teeth are more frequently missing in females than in males. Moreover, assuming genetic pleiotropy, the expression of the torus mandibularis, statural growth and the sex ratio (the ratio of the number of males to that of females) at birth and in the earlier stages of development are explained by this effect of the Y chromosome (Alvesalo 1985; Alvesalo 1997). The present results show that the effect of an extra X and Y chromosome on the promotion of dental development continues in the form of root dentin, which is influenced by cell proliferations, as was the formation of crown dentin before it. In accord, the tooth roots remain shorter than normal in individuals with a shortage of sex chromosome material. It is suggested that root formation may be affected by the same genes on the X and Y chromosomes that promote crown development.

In terms of population dental developmental standards (Haavikko 1970), the final size of the permanent tooth roots (excluding the third molars) becomes evident during a period beginning eight years after birth and continuing up to the age of 14 years, at least. Consequently, the present results indicate that the changes in tooth root lengths in all study groups become evident in final form within these time limits. Previous results regarding tooth crowns indicate that they reach their final size and shape between the ages of two months and eight years.

The possible effect of hormones as causative factors leading to changes in root growth in these groups of patients was considered. The hormone levels of 47,XYY males can be considered normal, but 47XXY males have insufficient testosterone production. 45,X/46,XX females with a shortage of sex chromosome material lack primary oestrogen. Because of the androgen insensitivity of 46,XY females, dental growth excesses in this group of individuals conceivably would develop independently of the possible influence of the androgens. All in all, it is difficult to explain that the differences in root lengths

54

between the individuals result from hormone production, hormone balance, hormone treatment or insensitivity to hormone.

It became clear that the effect of the Y chromosome on tooth root growth is greater than that of the X chromosome, and it is conceivable that this may lead to the sexual dimorphism observed in root length, men having longer roots than women. The location of the crown growth-promoting region is apparently on the long arm of the Y chromosome and the short arm of the X chromosome (Alvesalo & Chapelle 1981; Mayhall et al. 1991) as is also the location of the tooth root growth-promoting region, as suggested. It is of interest that the gene for amelogenin, which is the main protein component of the enamel matrix, has been located on either the long or short arm of the Y chromosome and on the short arm of the X chromosome (Lau et al. 1989).

Future work will be devoted to tooth root growth in 47,XXX, 48,XXXX, and 46,X,i(Xq) females, focusing on varying X chromosome constitutions. Another topic of interest will be heights of the tooth crowns.

7 Conclusions

The results pointed to longer tooth roots in humans due to additional sex chromosomes and shorter roots due to a shortage of sex chromosome material by comparison with the normal population. In 46,XY females, who are insensitive to androgens and phenotypically women, root size was considered similar to that of normal men.

It is also concluded that:

− final tooth root size changes in these groups of individuals apparently become evident during a period beginning at the age of eight years after birth and continuing at least up to the age of 14 years.

− the effect of the Y chromosome on root growth is greater than that of the X chromosome.

It is suggested that the genes on the X and Y chromosome that affect tooth crown growth are also expressed in the following root growth and may lead to sexual dimorphism in root length, men having longer roots than women.

References

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Alvesalo L (1985) Dental Growth in 47,XYY Males and in Conditions With Other Sex-Chromosome Anomalies. In: Sandberg AA (ed) The Y Chromosome, vol. 6, part B: Clinical Aspects of Y Chromosome Abnormalities. Alan R Liss, New York USA: 277-300.

Alvesalo L (1971) The influence of sex-chromosome genes on tooth size in man. A genetic and quantitative study. Suom Hammaslääk Toim 67(1): 3-54.

Alvesalo L & de la Chapelle A (1981) Tooth sizes in two males with deletions of the long arm of the Y-chromosome. Ann Hum Genet 45(Pt 1): 49-54.

Alvesalo L & Kari M (1977) Sizes of deciduous teeth in 47,XYY males. Am J Hum Genet 29(5): 486-489.

Alvesalo L & Laine T (1992) Occlusion in 47,XXY (Klinefelter syndrome) men. Am J Phys Anthropol 87(2): 161-165.

Alvesalo L, Nuutila M & Portin P (1975b) The cusp of Carabelli. Occurrence in first upper molars and evaluation of its heritability. Acta Odontol Scand 33(4): 191-197.

Alvesalo L, Osborne RH & Kari M (1975a) The 47,XYY male, Y chromosome, and tooth size. Am J Hum Genet 27(1): 53-61.

Alvesalo L & Portin P (1980) 47,XXY males: sex chromosomes and tooth size. Am J Hum Genet 32(6): 955-959.

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Original articles

I Lähdesmäki R, Alvesalo L (2004) Root lengths in 47,XYY males’ permanent teeth. J Dent Res 83: 771-775.

II Lähdesmäki R, Alvesalo L (2005) Root growth in the teeth of 46,XY females. Arch Oral Biol 50: 947-952.

III Lähdesmäki R, Alvesalo L (2006) Root growth in the permanent teeth of 45,X/46,XX females. Eur J Orthod 28: 339-344.

IV Lähdesmäki R, Alvesalo L. Root growth in the teeth of Klinefelter (47,XXY) men (manuscript).

I thank International & American Associations for Dental Research (I), Elsevier Ltd. (II), and Oxford University Press (III) for their permission to reprint the original papers in this thesis.

Original publications are not included in the electronic version of the dissertation.

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