2006 Wiley-Liss, Inc.

American Journal of Medical Genetics Part A 140A:2536 2546 (2006)

Research Review

Molecular Basis of Human Dentin Diseases

Mary MacDougall,1* Juan Dong,1 and Ana Carolina Acevedo2
1

Department of Oral Maxillofacial Surgery, Institute of Oral Health Research, School of Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 2 Departamento de Odontologia, Clinica de Anomalias Dentarias, Hospital Universitario, Faculdade de Ciencias da Saude, Universidade de Brasilia, Brasilia, Brazil
Received 1 May 2006; Accepted 20 May 2006

In recent years, substantial progress has been made regarding the molecular etiology of human structural tooth diseases that alter dentin matrix formation. These diseases have been classied into two major groups with subtypes: dentin dysplasia (DD) types I and II and dentinogenesis imperfecta (DGI) types IIII. Genetic linkage studies have identied the critical loci for DD-II, DGI-II, and DGI-II to human chromosome 4q21. Located within the common disease loci for these diseases is cluster of dentin/bone genes that includes osteopontin (OPN), bone sialoprotein (BSP), matrix extracellular phosphoglycoprotein (MEPE), dentin

matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP). To date, only mutations within dentin sialophosphoprotein have been associated with the pathogenesis of dentin diseases including DGI types-II and -III and DD-II. In this article, we overview the recent literature related to these dentin genetic diseases, their clinical features, and molecular pathogenesis. 2006 Wiley-Liss, Inc.

Key words: dentin dysplasia; dentinogenesis imperfecta; dentin sialophosphoprotein; SIBLINGS

How to cite this article: MacDougall M, Dong J, Acevedo AC. 2006. Molecular basis of human dentin diseases. Am J Med Genet Part A 140A:25362546.

INTRODUCTION

Tooth development, resulting from complex reciprocal interactions between oral epithelium and cranial neural crest-derived ectomesenchyme, has been classied into distinct stages: dental lamina, bud, cap, bell and crown [Thesleff, 2003]. During the transition from cap to bell stage, the cranial neural crest-derived dental papilla mesenchyme cells in contract with the basement membrane lining the developing tooth pulp chamber undergo cytodifferentiation in a process known as dentinogenesis. Dentinogenesis results in the formation of highly specialized cells termed odontoblasts that synthesized and secrete the dentin extracellular matrix (DECM) [see review Butler et al., 2003]. The DECM consists of two distinct layers: the non-mineralized predentin adjacent to the odontoblast cell bodies and the mineralized dentin located distally in contact with the overlaying enamel matrix. The odontoblasts are highly polarized cells with a long branched cellular process, termed odontoblastic cell process, that transverse the forming extracellular matrix. These cells secrete organic components similar to bone forming osteoblasts consisting of mostly type I

(approximately 86%), type I trimer, type III, type V, and type VI collagens and non-collagenous proteins (NCPS). The dentin/bone NCPs consist of osteonectin (OSN, also known as SPARC), osteocalcin (OC), and the family of small integrin-binding ligand, Nlinked glycoproteins (SIBLINGS). In the last decade, our understanding of the process of DECM formation and biomineralization has dramatically increased through the identication of major dentin extracellular matrix proteins, establishment of transgenic gene knock-out models, and the molecular analysis of human genetic diseases affecting dentin. Mainly, two approaches to identify disease-genes have been implemented: functional and positional-candidate cloning. Using a functional approach, dentin proteins have been cloned, their gene structure and chromosomal mapping

*Correspondence to: Mary MacDougall, Ph.D., Department of Oral Maxillofacial Surgery, University of Alabama at Birmingham School of Dentistry, Institute of Oral Health Research, 1530 3rd Avenue South SDB 702, Birmingham, AL 35294-0007. E-mail: MacDougall@uab.edu DOI 10.1002/ajmg.a.31359

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a HUMAN DENTIN DISEASES

2537

determined, and mutational analysis performed related to human tooth diseases. In the positionalcandidate approach, the disease locus is initially identied by genetic and physical mapping then candidate genes are identied by their physical localization with the critical disease locus. These candidate genes can be identied through physical map databases of genes and/or expressed sequence tags. In this article, we review recent literature related to the classications, clinical features, and molecular pathogenesis of heritable structural tooth diseases affecting dentin formation.
SIBLINGS

The major class of NCPs is the SIBLINGs consisting of osteopontin (OPN), matrix extracellular phosphoglycoprotein (MEPE), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), and dentin sialophosphoprotein (DSPP) [Fisher et al., 2001; MacDougall, 2003]. SIBLING proteins are secreted during the formation and mineralization of both dentin and bone but are also associated with other mineralized tissues such as cartilage and enamel. Furthermore, several reports have shown that SIBLINGS are

expressed in non-mineralized tissues such as kidney [Hudkins et al., 1999; Ogbureke and Fisher, 2005] and salivary glands [Ogbureke and Fisher, 2004]. The protein levels of BSP, OPN, DMP1, and DSPP found in dentin and bone are quantitatively very different [Qin et al., 2001]. In addition, in situ hybridization and immunohistochemistry studies have shown that the cellular expression proles and localization patterns of SIBLINGS within the ECM varies (Fig. 1). These proteins share similar features such as the presence of Arg-Gly-Asp (RGD) motif that mediates cell attachment/signaling via its reaction with cellsurface integrins and post-translational modications such as phosphorylation and glycosylation [Fisher et al., 2003; Qin et al., 2004]. Siblings also share common gene structure features including small non-rst exons, start codons contained in exon 2, the rst two amino acids encoded by exon 2, and large coding segments contained in the last exon.
Osteopontin (OPN) and Bone Sialoprotein (BSP)

OPN (also known as secreted phosphorprotein 1, SPP1) is a secreted 60-kDa phosphoprotein present in relatively large quantities in bone [Senger et al.,

FIG. 1. In situ hybridization showing the differential mRNA expression of DSPP, DMP1, and MEPE during developing tooth formation. Left panels are brighteld views while right panels are darkeld images. Probes are indicated as: DSPP, dentin sialophosphoprotein; DMP1, dentin matrix protein 1; and MEPE, matrix extracellular phosphoglycoprotein. Abbreviations: M1, rst molar; M2, second molar; I, incisor; and AvB, alveolar bone.

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2538

MACDOUGALL ET AL.

1979; Butler, 1989] but also as a component of dentin [Somerman et al., 1992; Papagerakis et al., 2002]. However, OPN is also expressed in a variety of other tissues and cells [Butler et al., 1996; Sodek et al., 2000]. Data from OPN null mice suggest that this protein may be a major inhibitory factor of mineralization [Boskey et al., 2002]. BSP is highly post-translationally modied protein that is an abundant non-collagenous component of the bone matrix [Fisher et al., 1990]. In situ hybridization and immunolocalization experiments have shown that BSP is expressed in rat and pig teeth during dentinogenesis [Chen et al., 1992, 1993]. Initially, OPN and BSP were seen as strong candidates for dentinogenesis imperfecta type II (DGI-II) since linkage analysis using two large families demonstrated no recombination events with these markers and the disease. However, mutational analysis of both SPP1 (OPN) and IBSP (BSP) failed to reveal any mutations associated with the pathogenesis of DGI type II [Crosby et al., 1995, 1996].
Matrix Extracellular Phosphoglycoprotein (MEPE)

dentin/bone gene cluster locating MEPE/OF45 between BSP and SPP1 through gene and STRP content mapping. Initial studies have demonstrated that like other SIBLINGs, MEPE/OF45 is expressed by odontoblasts during tooth formation [MacDougall et al., 2002]. Characterization of the MEPE/OF45 gene structure based on human, rat, and macaca transcripts shows that it organized into at least ve exons and four introns. To date, no mutations within MEPE/ OF45 have been reported associated with any dentin disease. Targeted disruption of MEPE results in increased bone formation and bone mass [Gowen et al., 2003]. Cancellous bone histomorphometry revealed that the increased bone mass in the knockout mice was the result of an increase in the number of osteoblast and osteoclast activity with unaltered osteoclast number. These results suggest an inhibitory role for MEPE in bone formation in mouse.
Dentin Matrix Protein 1 (DMP1)

Recently, MEPE a new bone matrix protein has been independently cloned from human and rat tissues. The human clone, matrix MEPE, was isolated from a human oncogenic hypophosphatemic osteomalacia (OHO) tumor cDNA library [Rowe et al., 2001], while the rat clone, termed osteogenic factor 45 kDa (OF 45, also termed osteoregulin), was isolated from bone by subtractive hybridization based upon high expression in bone marrowderived osteoblasts [Petersen et al., 2001]. The human MEPE 1,989-bp full-length cDNA clone encodes a predicted 525 bp rich in asparate, serine and glutatmate residues (26%) containing a 17 amino acid signal peptide. MEPE contains two N-glycosylation motifs (NNST and NNSR), a glycosaminoglycan attachment site (SGDG), an RGD cell attachment motif, several predicted phosphorylation motifs, and N-myristoylation sites. MEPE has been shown to have sequence homology to DSPP, DMP1, and OPN as identied using the TREMBL database. An acidic serine-aspartate-rich motif (DDSSESSDSGSSSESD) that occurs once at the COOH terminal has 80% sequence homology and 93% physiochemical similarity with a reoccurring motif in DSPP (SDSSD SSDSSSSSDSS). Two other DSPP sequence motifs (DSSDSSDSNSSSDS and DSSDSSDSSNSSDS) have 80% homology while OPN (DDSHQSDESHHSDESD) and DMP1 (SSRRRDDSSESSDSGSSSESDG) motifs have approximately 60% homology. Human chromosomal mapping studies place MEPE gene on human 4q21q23 between EST markers D4S2785 (WI-6336) and D4S2844 [Rowe et al., 2001]. Rened mapping performed in our laboratory has further dened the locus in the

DMP1 (formerly AG-1) is an acidic, phosphorylated protein rst identied by cDNA cloning from a rat pulp library. Initial analysis by Northern blot hybridization of a variety of rat tissues and in situ hybridization studies of developing rat teeth detected DMP1 mRNA transcripts only in odontoblasts with transient expression in ameloblasts [George et al., 1993, 1994]. This data suggested DMP1 expression was restricted to the DECM. However, more extensive in situ hybridization studies have shown that the protein is expressed at high levels in developing bone and at lower levels in cartilage and brain [DSouza et al., 1997; Hirst et al., 1997; Feng et al., 2003]. Analysis mouse and human cDNA clones identied a novel 45-bp segment encoding 15 amino acids representing the full length transcript suggesting alternative giving rise to two different isoforms [Hirst et al., 1997; MacDougall, 1998]. DMP1 is suggested to bind tightly to hydroxyapatite, and mediate cell attachment through a RGD domain [Kulkarni et al., 2000]. DMP1 has been mapped to human chromosome 4 [Aplin et al., 1995] at band 4q21.2q21.3 by FISH [MacDougall, 1998]. DMP1 has been tightly linked to DGI-II, Zmax 11.0 (y 0.001), suggesting this gene is a strong candidate for DGI-II, DGI-III, and DD-II [Aplin et al., 1995]. However, mutational analysis has excluded this gene from a causative role in the pathogenesis of DGI-II within the families studied [Hirst et al., 1997]. More recently, it was shown that postnatal Dmp-1 null mice develop a tooth phenotype characterized by incomplete maturation of predentin into dentin, enlarged pulp chambers, increased width of predentin zone with reduced dentin wall, and hypomineralization [Ye et al., 2004].

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a HUMAN DENTIN DISEASES

2539

Dentin Sialophosphoprotein (DSPP)

Among the non-collagenous proteins, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are the major proteins and are believed to play a crucial role in dentinogenesis. These two proteins are derived from the cleavage of a 940amino acid polypeptide, DSPP [MacDougall et al., 1997a]. The structural organization of the mouse DSPP gene has conrmed that both dentin proteins are encoded by one gene, with a continuous open reading frame. The DSPP gene in all species studied to date is organized into ve exons and four introns [MacDougall et al., 1997a]. In situ hybridization studies indicate DSPP expression is conned to differentiating odontoblasts, with transient expression by presecretory ameloblasts [DSouza et al., 1992, 1997; Begue-Kirn et al., 1998] (Fig. 1). Recently, however, low levels of DSPP have been observed in the ear and in bone [Xiao et al., 2001; Qin et al., 2003]. DSP, the amino-terminal domain of DSPP, is a sialic acid rich and glycosylated protein that shares similarities with the other sialoproteins, including BSP, DMP-1, and OPN. The COOH-terminal domain DPP is a highly phosphorylated protein with repeats of aspartic acid and phosphoserine. DPP is thought to play a key role in the nucleation of hydroxyapatite formation during dentin calcication [Veis, 1993; George et al., 1996; Butler, 1998]. Human DSPP has been mapped by FISH to 4q21.3, within an overlapping regions for DGI-II, DGI-III, and DD type II (DD-II) loci [MacDougall et al., 1997b, 1998]. Dspp-null mice have shown tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure [Sreenath et al., 2003].
DENTIN STRUCTURAL GENETIC DISEASES

dened by markers GATA62A11and D4S1563 [Aplin et al., 1999]. Linkage analyses of DGI-III showed no recombination events with DMP1, maximum lod score of 4.86, mapping the critical locus to 4q21.1 21.3 within a 6.6 cM region that overlaps the critical DGI Type II region [MacDougall et al., 1999]. Dean et al. [1997] established linkage for DD Type II with a maximum two-point lod score of 4.2, y 0.0 within a 14.1 cM region of human 4q21 dened by markers SPP1 and D4S2691. Therefore, the DD-II locus overlaps both the DGI Type II and III loci. These data suggest that all three dentin diseases are allelic (Fig. 2) [MacDougall, 2003].
PHENOTYPIC CHARACTERISTICS Dentin Dysplasia Type I (DD-I)

DD-I also known as Rootless teeth (OMIM 125400) is a rare autosomal dominant genetic disease causing incomplete tooth formation that results in premature exfoliation of both the primary and permanent dentitions. The enamel and coronal dentin are well formed, but the radicular dentin lacks histological organization and subsequently is shortened dramatically. The patients present with normal appearing crowns in both deciduous and permanent dentitions, short, conical, or absent roots, and partial or full obliteration of the crown pulp chamber except for a thin, crescent-shaped pulp remnant. Histologically, the mantle dentin and the majority of the coronal dentin are unaffected. [Shields et al., 1973; Witkop, 1975; OCarroll and Duncan, 1994]. However, associated with the pulpal remnant, abnormal dentin is seen within large pulp stones with calcied tubular dentin and atypical osteodentin [Melnick et al., 1980].
Dentin Dysplasia Type II (DD-II)

Genetic diseases that primarily affect dentin formation are known as heritable dentin structural diseases. To date, all reported dentin diseases exhibit an autosomal dominant pattern of inheritance and are classied into two major groups with further subclassications: Dentin Dysplasia with types I and II and Dentinogenesis Imperfecta with types IIII [Shields et al., 1973; Waltimo, 1996].
Chromosomal Mapping

Initial linkage studies placed the DGI-II gene on human chromosome 4q near the group-specic component gene (Gc) [Ball et al., 1982] and interferon-inducible cytokine IP-10 gene [Crall, 1989; Crall et al., 1992]. More precise mapping placed the locus at 4q21q23 in a 6.6 cM region dened by markers D4S2691 and D4S2692 [Crosby et al., 1995a,b] that was further rened to a 2 cM region

DD II (OMIM 125420) is a rare autosomal dominant condition that affects mainly the deciduous dentition. Deciduous teeth are opalescent and have a grayish or brownish discoloration similar to that seen in DGI while permanent teeth have normal clinically coloration. Radiographically, the primary teeth show pulp obliteration whereas the permanent teeth have a thistle-tube pulp chamber with pulp stones. The roots of both dentitions appear normal [Shields et al., 1973; Witkop, 1975; OCarroll et al., 1991]. Histologically, the dentin of DD II deciduous teeth is highly disorganized with few dentinal tubules [Melnick et al., 1977].
Dentinogenesis Imperfecta (DGI)

DGI is the most common group of inherited dentin defects. According to the Shields classication system, which is based on clinical and radiographic features, this disease has been divided into three

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2540

MACDOUGALL ET AL.

FIG. 2. Schematic diagram summarizing the relationship of the cytogenetic and linkage maps of human chromosome 4 to the overlapping critical loci for the dental diseases DGI Types II and III, and DD Type II. The region between markers D4S2691 and SSP1 contains the SIBLING gene cluster. Abbreviations: p, short arm; q, long arm; cM, centiMorgan; and kb, kilobases.

subgroups: Types I, II, and III. DGI type I is associated with osteogenesis imperfecta (brittle bone disease) caused by heterogeneous mutations in either COL1A1 or COL1A2, the genes that encode type 1 collagen chains. Collagen type I is the major structural protein found in both dentin and bone ECMs [Byers, 2000]. In general, DGI teeth show marked discoloration and attrition in both the deciduous and permanent dentition. Radiographically, both dentitions show short constricted roots with progressive pulpal obliteration. The degree of expressivity is variable, even within a single individual patient, ranging from total pulpal obliteration to normal-appearing dentin [Shields et al., 1973]. DGI type II, also called opalescent dentin (DGI-II, DGI1; OMIM 125490), is a more common genetic tooth disorder, with an estimated incidence in the United States between 1:6,000 and 1:8,000 [Witkop, 1957]. Both deciduous and permanent teeth are affected, appearing discolored (yellow, amber brown, or bluish gray) and translucent (Fig. 3A,B) The enamel tends to chip from the underlying softer

dentin and variable degrees of attrition can be observed, especially in the permanent dentition. Radiographically, the teeth have bulbous shaped crowns due to cervical constrictions at the root junction. The roots often appear smaller and more narrow than normal. The pulp chambers and root canals are usually obliterated (Fig. 3C,D) [Witkop and Rao, 1971; Shields et al., 1973; Witkop, 1975]. The histopathological analysis shows that the initially formed mantle dentin is normal. However, the dentinal tubules of the circumpulpal dentin are decreased displaying a disorganized distribution with smaller diameters [Kerebel, 1975] (Fig. 3E). The inorganic phase of DGI type II dentin is also altered with a decrease in the number of crystallites and a signicant decrease in the total mineral content as compared with normal dentin [Kerebel et al., 1981]. More recently, using high resolution synchrotron radiation computed tomography and small angle X-ray scattering, a mineral decrease of 33% in DGI type II dentin was shown as compared with normal dentin. Furthermore, it was suggested that

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a HUMAN DENTIN DISEASES

2541

FIG. 3. Clinical, radiographic, and histological dental manifestations of typical DGI-II patients. Panel A, clinical photograph showing the yellow-brownish discoloration of deciduous teeth with mild attrition observed in the upper and lower anterior teeth. Enamel opacities are also seen in all upper teeth as well as in lower canines and molars. Panel B, clinical photograph showing a lower occlusal view of deciduous teeth with attrition of the lower anterior teeth. Panels C, D are panoramic radiographs showing deciduous (C) and mixed dentition (D). Erupted deciduous and permanent teeth show bulbous crown appearance due to cervical crown constriction (arrowheads). In the developing permanent teeth, pulp chambers and root canals are evident (arrow). However, in the erupted permanent teeth, pulp chambers are obliterated (asterisks) and root canals are dramatically reduced. Panel E, photograph of a molar ground section showing aberrant crown circumpulpal dentinal tubules (arrow) and a reduction of the root dentinal tubules (arrowhead). Abbreviations: E, enamel; and D, dentin.

intrabrillar mineral may be absent in DGI type II dentin [Kinney et al., 2001]. DGI Type III (DGI-III, OMIM 125500) affects the Brandywine isolate a tri-racial subpopulation consisting of Native American Indians, African Americans, and Caucasians of European decent located initially in southern Maryland. This population has the highest incidence of any dental genetic disease estimated at 1:15 [Hursey et al., 1956; Witkop et al., 1966]. The clinical manifestations of DGI-III affect both dentition but differ from DGI-II in the presence of multiple pulp exposures, normal nonmineralized pulp chambers and canals and enamel pitting defects. The teeth of patients with DGI-III are referred to as shell teeth, based on the limited dentin mineralization after initial mantle dentin formation. Radiographically, the pulp cavities in these teeth appear as enlarged pulp chambers along with a high incidence of pulp exposures [Shields et al., 1973; Levin et al., 1983].

Mutations Associated With Dentin Diseases

To date, only DSPP mutations have been associated with the pathogenesis of dentin diseases including DGI-II and -III and DD-II (Table I). At present, nine DSPP mutations have been reported causing DD-II [Rajpar et al., 2002], DGI-II [Xiao et al., 2001; Zhang et al., 2001; Kim et al., 2004, 2005; Malmgren et al., 2004], DGI-II with progressive hearing loss [Xiao et al., 2001] and DGI-III [Dong et al., 2005]. Interestingly, only in the case of DGI-III has a mutation within the DPP domain of DSPP been identied [Dong et al., 2005]. Although the other SIBLINGs are potential candidate genes for inherited dentin diseases based on their chromosomal mapping within the dentin/bone gene cluster, no disease-causing mutations have yet been identied. The rst mutations associated with DGI were reported in four independent Chinese families [Xiao et al., 2001; Zhang et al., 2001]. Zhang and

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2542

MACDOUGALL ET AL. TABLE I. Identied DSPP Mutations in Dentinogenesis Imperfecta (DGI) and Dentin Dysplasia (DD) Families

Races/country Missense mutations Not reported Chinese/China Central American/Sweden Chinese/China Caucasian/US Korean/Korean Caucasian/Sweden Nonsense Mutation Chinese/China Splicing site mutations Chinese/China Korean/Korean Deletion/insertion Brandywine isolate/US

cDNA (NM_014208) 16 T > G 49 C > A 44 C > T 52 G > T 52 G > T 52 G > T 202 A > T 133 C > T IVS 3 1 IVS 2-3 3595 ins18 bp 3479del36bp

Amino acid Tyr 6 Asp Pro 17 Thr Ala 15Val Val 18 Phe Val 18 Phe Val 18 Phe Arg 68 Trp Gln 45 stop ? ? Truncation

Sequences TAT > GAT CCA > ACA GCC > GTC GTT > TTT GTT > TTT GTT > TTT AGG > TGG CAG > TAG TACAGg/a cag > gag Ins 18 bp Del 36 bp

Exon/ Intron Exon 2 Exon 2 Exon 2 Exon 3 Exon 3 Exon 3 Exon 4 Exon 3

Phenotype DD-II DGI-II hearing loss DGI-II DGI-II hearing loss DGI-II DGI-III DGI-II DGI-II

Reference Rajpar et al. [2002] Xiao et al. [2001] Malmgren et al. [2004] Xiao et al. [2001] Kim et al. [2005] Kim et al. [2005] Malmgren et al. [2004] Zhang et al. [2001]

Intron 3 1 DGI-II Xiao et al. [2001] Intron 2 3 DGI-II, one affected with Kim et al. [2004] mild hearing loss Exon 5 DGI-III Dong et al. [2005]

co-workers, 2001 identied a DGI-II family a nonsense mutation (c.133C ! T) in exon 3 changing Gln 45 to a stop codon dramatically truncating the DSPP protein eliminating the majority of the DSP domain and the entire DPP domain. In this family, the phenotype would likely be generated by a haploinsufancy. Xiao et al. [2001] studied three different DGI-II families, one presenting with only DGI-II and two presenting with DGI-II and progressive sensorineural high-frequency hearing loss (DFNA 39). In the family presenting with DGI-II, only a G ! A mutation was identied in the donor-splicing site of intron 3 that would potentially cause the skipping of exon 3. In the other two families with DGI-II and progressive hearing loss, different single nucleotide changes were found, c.49C ! A and c.G52 ! T, causing changes in adjacent amino acids, Pro17Thr and Val18Phe, encoded in exon 2 and exon 3. Due to the association with hearing problems, these authors demonstrated for the rst time that DSPP is expression in the mouse inner ear by RT-PCR analysis. More recently, the same missense mutation in exon 3 (c.52G ! T, Val18Phe) was reported in two additional families, one Korean and one Caucasian family [Kim et al., 2005]. In the Korean family, only a single person was affected, the 25-month old female proband. Kim et al. [2005] made a diagnosis of DGIIII based on the presence of wide pulp chambers, pulpal exposures, and periapical inammation. Since no mutations were identied in other family members, they suggested that the mutation had arisen spontaneously in that family. However, since the only affected individual was very young, caution is warranted in the precise diagnosis especially as related to DGI-II since the phenotype is progressive. A long-term follow up of the patient would be important and justied. In the three generation,

Caucasian family, the phenotype was consistent with DGI-II. No hearing loss was detected in either family. Other missense DSPP mutations were identied in two families: one of Swedish decent and one originally from Central America living in Sweden [Malmgren et al., 2004]. Both families exhibited a DGI-II phenotype with no detectable progressive hearing loss. The two families presented different missense mutations, c.202A ! T and c.44C ! T, causing changes in Arg68Trp and Ala15Val, in exon 4 and exon 2, respectively. Furthermore, a splice acceptor mutation (IVS2-3C ! G) in the third from the last nucleotide from intron 2 and changed its sequence from CAG to GAG was shown in a Korean DGI II family with one affected family member that had a mild hearing loss [Kim et al., 2004]. A mutation (c.16T ! G,Tyr6Asp) within the signal peptide of DSSP has been shown in a family diagnosed with DD-II [Rajpar et al., 2002]. More recently, a rare compound mutation involving a 36-bp deletion and 18-bp insertion within exon 5 of the DSPP gene has been identied in a family with dentinogenesis imperfecta type III (DGIIII) [Dong et al., 2005]. The 36-bp deletion resulted in an in frame deletion of 12 codons (SDSSDSSDSSDS 1160_1171del) causing a shortening of a (DSS)16 repetitive segment to (DSS)14 near the COOH terminus. The 18-bp insertion represented an 18-bp duplication causing a six amino acid insertion (SDSSDS1198_1199ins) between amino acid 1,198 and 1,199. This insertion augments an additional (DSS)4 repeat to (DSS)6 within the DPP coding region. The net result of this compound mutation was a shortening of the DPP domain near the COOH region by six amino acids with no new restriction enzyme sites created. Associated with DGI-III, the identied mutations both involve a highly repetitive

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a HUMAN DENTIN DISEASES

2543
Malmgren et al. [1988, 2004] Zhang et al. [2001] Xiao et al. [2001] Kim et al. [2004] MacDougall et al. [1999]; Dong et al. [2005] NR (deciduous) NR NR NR NR NR NR NR NR NR Dysplastic dentin NR NR NR NR DGI II DGI II DGI II DGI III DGI II
D, deciduous teeth; P, permanent teeth; NR, not reported.

sequence of DNA. In fact, the inserted segment (18 bp) that is duplicated is found 10 times within the DPP domain. The mechanisms of this mutation may be the similar with neurological diseases with trinucleotide repeat mutations [Cummings and Zoghbi, 2000] since a portion of the mutated sequences (gcagtgacagca) is duplicated 53 times in DSPP and represents 21% of the entire DPP domain. This and other highly repeated sequences in DSPP may have an important function during evolution in gene duplication and diversication of the dentin/ bone SIBLING gene family. DGI-III is considered a distinct clinical entity based on the presence of viable pulp chambers and the appearance of shell teeth in dental X-rays in contrast with DGI-II and DD-II. Since both DGI-II and DD-II are associated with alternations in DSP while DGI-III is caused by alterations in DPP, these diseases provide insight as to the differing biological functions of these major tooth extracellular matrix proteins. The identied mutations associated with DGI-II all occur within DSP encoding exons 2, 3, and 4 resulting in a truncation of the protein that may have a dominant negative effect. The missense mutation (Tyr6Asp) within the hydrophobic signal peptide found in DD-II, causing a failure of translocation of the protein to the endoplasmic reticulum, likely results in haploinsufancy decreasing levels of both DSP and DPP [Rajpar et al., 2002]. With the identied DGI-III mutation in this family only DPP levels would be altered with normal levels of DSP. Therefore, the function of DSP could be associated with synthesis and rate of dentin matrix formation and the conversion of predentin to mineralized dentin. Abnormal or altered levels of DSP seen in DGI-II and DD-II would cause the pulp chamber obliteration. DPP alternations or abnormal levels associated with all three diseases would be correlated with abnormal dentin matrix mineralization and discoloration of the teeth. In order to make more accurate diagnoses related to dentin diseases, a clear understanding of the genotype/phenotype correlation is necessary. Therefore, it is critical that our database be based on many more families with detailed longitudinal phenotypic characterizations of their dentitions since these are progressive diseases. When we analyze retrospectively the descriptions of the families with identied mutations (Table II), we can see that that the phenotypic details that are reported are inconsistent limiting the comparisons between different families. Malmgren et al. [2004] presented the most comprehensive clinical description of two families showing long-term clinical and radiographic followups as well as histopathological analysis. Since the pulp chamber and canals obliteration is a progressive process, the radiographic follow-ups at different ages are very useful in determining the extent of pulpal chambers and canals. Special attention has to

Histopathology TABLE II. Dental Phenopytic Characterization in Dentinogenesis Imperfecta and Dentin Dysplasia Families Periapical lesions

NR NR (permanent) NR (Deciduous) NR NR (Deciduous) NR D/P NR D/P NR NR

Pulp stones

Pulp chamber/ canal obliterations

Abnormal constriction Coronal/ Radicular junction

Tooth discoloration

Dentition affected

NR

D/P 202 A > T

NR (anterior teeth) NR D D DGI II DGI III DGI II 52 G > T 52 G > T 52 G > T

Attrition

NR

NR (deciduous)

Wide pulp chambers (shell teeth)

NR

NR

NR

NR NR Dysplastic dentin NR NR NR

Rajpar et al. [2002] Xiao et al. [2001] Malmgren et al. [2004] Xiao et al. [2001] Kim et al. [2005] Kim et al. [2005]

References

Mutations cDNA (NM_014208)

Diagnosis

DD II DGI II DGI II

133 C > T IVS 3 1 IVS 2 - 3 3595 ins18bp 3479del36bp

16 T > G 49 C > A 44 C > T

D/P NR D/P D/P

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2544

MACDOUGALL ET AL.

be given to the diagnosis of young family members. Again, Malmgrem et al. [1998] reported a sixgeneration family with two young patients (16 months and 2 years old) with large pulp chambers, and periapical radiolucencies, at older ages, the permanent teeth of these patients showed pulpal obliterations. Also, Kim et al. [2005] reported a DSSP mutation in a 22 months old patient, with wide pulp chambers, multiple pulp exposures, and radiolucencies, diagnosed with DGI-III. However, since she was the only affected family member, it is not possible to compare the radiographic manifestations in older affected family members. Likely, in this young patient, pulpal obliterations will occur with age. The relatively limited current literature related to the correlation of genotype and phenotype for dentin diseases demonstrates the critical need for a standardized basic set of clinical features that need to be documented at multiple time points (years) to capture the complete clinical prole of these disease. This includes a complete physical examination focusing on potential skeletal problems as well as in progressive hearing loss. Some DGI-I families can have very mild skeletal features such as joint pain and hyperextensibility with practically no bone fractures [Pallos et al., 2001] leading to a misdiagnosis of DGI-II and misdirecting molecular studies (DSPP mutational analysis vs. COL1A1 or COL1A2). Studies of a larger number of families with welldened phenotypes are necessary in order to clarify the extent of which these various dentin diseases represent phenotypic variation of a single disease. At present, the Val18Phe mutation, is the most common missense mutation identied in the DSSP gene, present in families with diversity ethnic backgrounds. This mutation has been identied in individuals affected with only DGI-II, DGI-II with sensorineural hearing loss and DGI-III. Based on the emerging literature, patients with inherited dentin defects should be initial screened for potential mutations within DSPP especially exons 24.
ACKNOWLEDGMENTS

We thank the many families that participated in our genetic studies. The staff of the Dental Anomalies Clinic (University of Brasilia, Brazil) who collaborate in the diagnosis and treatment of the patients. We also acknowledge Dr. J. Gluhak-Heinrich for her performance of the in situ hybridization studies.
REFERENCES
Aplin HM, Hirst KL, Crosby AH, Dixon MJ. 1995. Mapping of the human dentin matrix acidic phosphoprotein gene (DMP1) to the dentinogenesis imperfecta type II critical region at chromosome 4q21. Genomics 30:347349.

Ball SP, Cook PJL, Mars M, Buckton KE. 1982. Linkage between dentinogenesis imperfecta and Gc Ann. Hum Genet 46:35 40. Begue-Kirn C, Krebsbach PH, Bartlett JD, Butler WT. 1998. Dentin sialoprotein, dentin phosphoprotein, enamelysin and ameloblastin: Tooth-specic molecules that are distinctively expressed during murine dental differentiation. Eur J Oral Sci 106:963970. Boskey AL, Spevak L, Paschalis E, Doly SB, McKee MD. 2002. Osteopontin deciency increases mineral content and mineral crystallinity in mouse bone. Calcif Tissue Int 71:145154. Butler WT. 1989. The nature and signicance of osteopontin. Connect Tissue Res 23:123136. Butler WT. 1998. Dentin matrix proteins. Eur J Oral Sci 106:204 210. Butler WT, Ridall AL, McKee MD. 1996. Osteopontin. In: Bilezikian JP, Raisz LG, Rodan GA, editors. Principles of Bone Biology. New York: Academic. p 167181. Butler WT, Brunn JC, Qin C. 2003. Dentin extracellular matrix (ECM) proteins: Comparison to bone ECM and contribution to dynamics of dentinogenesis. Connect Tissue Res 44:171 178. Byers PH. 2000. Collagens: Building blocks at the end of the development line. Clin Genet 58:270279. Chen J, Shapiro HS, Sodek J. 1992. Developmental expression of bone sialoprotein mRNA in rat mineralised connective tissues. J Bone Miner Res 8:987997. Chen J, McCulloch CAG, Sodek J. 1993. Bone sialoprotein in developing porcine dental tissues: Cellular expression and comparison of tissue localization with osteopontin and osteonectin. Arch Oral Biol 38:241249. Crall MG. 1989. Genetic marker study of dentinogenesis imperfecta. Masters Thesis, Ohio State University, Columbus. Crall MG, Schuler CF, Buetow KH, Murray JC. 1992. Multipoint linkage analysis of dentinogenesis imperfecta (DGI) on 4q. Proc Finn Dent Soc 88:286293. Crosby AH, Edwards SJ, Murray JC, Dixon MJ. 1995. Genomic organization of the human osteopontin gene: Exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II. Genomics 27:155160. Crosby AH, Lyu MS, Lin K, McBride OW, Kerr JM, Aplin HM, Fisher LW, Young MF, Kozak CA, Dixon MJ. 1996. Mapping of the human and mouse bone sialoprotein and osteopontin loci. Mamm Genome 7:149151. Cummings CJ, Zoghbi HY. 2000. Trinucleotide repeats: Mechanisms and pathophysiology. Annu Rev Genomics Hum Genet 1:281328. DSouza RN, Bronckers AL, Happonen RP, Doga DA, FarachCarson MC, Butler WT. 1992. Developmental expression of a 53 KD dentin sialoprotein in rat tooth organs. J Histochem Cytochem 40:359366. DSouza RN, Cavender A, Sunavala G, Alvarez J, Ohshima T, Kulkarni AB, MacDougall M. 1997. Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo. J Bone Miner Res 12:20402049. Dean JA, Hartseld JK Jr, Wright JT, Hart TC. 1997. Dentin dysplasia, type II linkage to chromosome 4q. J Craniofac Genet Dev Biol 17:172177. Dong J, Gu T, Jeffords L, MacDougall M. 2005. Dentin phosphoprotein compound mutation in dentin sialophosphoprotein causes dentinogenesis imperfecta type III. Am J Med Genet Part A 132A:305309. Feng JQ, Huang H, Lu Y, Ye L, Xie Y, Tsutsui TW, Kuneida T, Castranio T, Scotte G, Bonewald LB, Mishima Y. 2003. The Dentin matrix protein 1 (Dmp1) is specically expressed in mineralized, but not soft, tissues during development. J Dent Res 82:776780. Fisher LW, Fedarko NS. 2003. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins. Connect Tissue Res 44:3340.

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a HUMAN DENTIN DISEASES

2545

Fisher LW, McBride OW, Termine JD, Young MF. 1990. Human bone sialoprotein. Deduced protein sequence and chromosomal localization. J Biol Chem 265:23472351. Fisher LW, Torchia DA, Fohr B, Young MF, Fedarko NS. 2001. Flexible structures of SIBLING proteins, bone sialoprotein, andosteopontin. Biochem Biophys Res Commun 280:460 465. George A, Sabsay B, Simonian PA, Veis A. 1993. Characterizationof a novel dentin matrix acidic phosphoprotein. Implications forinduction of biomineralization. J Biol Chem 268:12624 12630. George A, Gui J, Jenkins NA, Gilbert DJ, Copeland NG, Veis A. 1994. In situ localization and chromosomal mapping of theAg1(DMP1) gene. J Histochem Cytochem 42:15271531. George A, Bannon L, Sabsay B, Dillon JW, Malone J, Veis A, Jenkins NA, Gilbert DJ, Copeland NG. 1996. The carboxylterminal domain of phosphophoryn contains unique extended triplet amino acid repeat sequences forming ordered carboxyl-phosphate interaction ridges that may be essential in the biomineralization process. J Biol Chem 271:3286932873. Gowen LC, Petersen DN, Mansolf AL, Qi H, Stock JL, Tkalcevic GT, Simmons HA, Crawford DT, Chidsey-Frink KL, Ke HZ, McNeish JD, Brown TA. 2003. Targeted disruption of the osteoblast/osteocyte factor 45 gene (OF45) results in increased bone formation and bone mass. J Biol Chem 17:19982007. Hirst KL, Simmons D, Feng J, Aplin H, Dixon M, Mac-Dougall M. 1997. Elucidation of the sequence and the genomicorganization of the human dentin matrix acidic phosphoprotein 1(DMP1) gene: Exclusion of the locus from a causative role in the pathogenesis of dentinogenesis imperfecta type II. Genomics 42:3845. Hudkins KL, Giachelli CM, Cui Y, Couser WG, Johnson RJ, Alpers CE. 1999. Osteopontin expression in fetal and mature human kidney. J Am Soc Nephro 10:14441457. Hursey RJ, Witkop CJ Jr, Miklashek D, Sackett LM. 1956. Dentinogenesis imperfecta in a racial isolate with multiple hereditary defects. Oral Surg 9:641658. Kerebel B. 1975. Dentinogenesis imperfecta: A structural and ultrastructural study. SSO Schweiz Monatsschr Zahnheilkd 85:12641281. Kerebel B, Daculsi G, Menenteau J, Kerebel LM. 1981. The inorganic phase in dentinogenesis imperfecta. J Dent Res 60:16551660. Kim JW, Nam SH, Jang KT, Lee SH, Kim CC, Hu JC, Hahn SH, Simmer JP. 2004. A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II. Hum Genet 115:248254. Kim JW, Hu JC, Lee JI, Moon SK, Kim YJ, Jang KT, Lee SH, Kim CC, Hahn SH, Simmer JP. 2005. Mutational hot spot in the DSPP gene causing dentinogenesis imperfecta type II. Hum Genet 116:186191. Kinney JH, Pople JA, Driesen CH, Breunig TM, Marshall GW, Marshall SJ. 2001. Intrabrillar mineral may be absent in dentinogenesis imperfecta type II (DI-II). J Dent Res 80:1555 1559. Kulkarni GV, Chen B, Malone JP, Narayanan AS, George A. 2000. Promotion of selective cell attachment by the RGD sequence in dentine matrix protein 1. Arch Oral Biol 45:475484. Levin LS, Leaf SH, Jelmini RJ, Rose JJ, Rosenbaum KN. 1983. Dentinogenesis imperfecta in the Brandywine isolate (DI type III): Clinical, radiologic, and scanning electron microscopic studies of the dentition. Oral Surg Oral Med Oral Path 56:267 274. MacDougall M. 1998. Rened mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci. Europ J Oral Sci 106:227233. MacDougall M. 2003. Dental structural diseases mapping to human chromosome 4q21. Connect Tissue Res 44:285291.

MacDougall M, Zeichner-David M, Slavkin HC. 1985. Production and characterization of antibodies against murine dentine phosphoprotein. Biochem J 232:493500. MacDougall M, Simmons D, Luan X, Nydegger J, Feng J, Gu TT. 1997a. Dentin phosphoprotein and dentin sialoprotein are cleavage products expressed from a single transcript coded by a gene on human chromosome 4: Dentin phosphoprotein DNA sequence determination. J Biol Chem 272:835842. MacDougall M, Simmons D, Luan X, Gu TT, DuPont BR. 1997b. Assignment of dentin sialophosphoprotein (DSPP) to the critical DG12 locus on human chromosome 4 band q21.3 by in situ hybridization. Cytogenet Cell Genet 79:121122. MacDougall M, Jeffords LG, Gu TT, Knight CB, Frei G, Reus BE, Otterud B, Leppert M, Leach RJ. 1999. Genetic linkage of the dentinogenesis imperfecta type III locus to chromosome 4q. J Dent Res 78:12771282. MacDougall M, Simmons D, Gu TT, Dong J. 2002. MEPE/OF45 a new dentin/bone matrix protein and candidate gene for dentin diseases mapping to 4q21. Connect Tiss Res 43:373 375. Malmgren B, Lundberg M, Lindskog S. 1988. Dentinogenesisimperfecta in a six-generation family. A clinical, radiographic and histologic comparison of two branches through three generations. Swed Dent J 12:7384. Malmgren B, Lindskog S, Elgadi A, Norgren S. 2004. Clinical, histopathologic, and genetic investigation in two large families with dentinogenesis imperfecta type II. Hum Genet 114:491498. Melnick M, Eastman JR, Goldblatt LI, Michaud M, Bixler D. 1977. Dentin dysplasia, type II: A rare autosomal dominant disorder. Oral Surg 44:592599. Melnick M, Levin LS, Brady J. 1980. Dentin dysplasia type I: A scanning electron microscopic analysis of the primary dentition. Oral Surg 50:335339. OCarroll MK, Duncan WK. 1994. Dentin dysplasia type I. Radiologic and genetic perspectives in a six-generation family. Oral Surg Oral Med Oral Pathol 78:375381. OCarroll MK, Duncan WK, Perkins TM. 1991. Dentin dysplasia: Review of the literature and a proposed subclassication based on radiographic ndings. Oral Surg Oral Med Oral Pathol 72:119125. Ogbureke KU, Fisher LW. 2004. Expression of SIBLINGs and their partner MMPs in salivary glands. J Dent Res 83:664 670. Ogbureke KU, Fisher LW. 2005. Renal expression of SIBLING proteins and their partner matrix metalloproteinases (MMPs). Kidney Int 68:155166. Pallos D, Hart PS, Cortelli JR, Vian S, Wright JT, Korkko J, Brunoni D, Hart TC. 2001. Novel COL1A1 mutation (G559C) [correction of G599C] associated with mild osteogenesis imperfecta and dentinogenesis imperfecta. Oral Biol 46:459470. Papagerakis P, Berdel A, Mesbah M, Peuchmaur M, Malaval L, Nydegger J, Simmer J, MacDougall M. 2002. Investigation of ostoeocalcin, osteonectin, and dentin sialophosphrotein in developing human teeth. Bone 30:377385. Petersen DN, Tkalcevic GT, Vai AL, Rivera-Gonzalez R, Brown TA. 2001. Identication of osteoblast/osteocytes factor 45 (OF45) a bone specic cDNA encoding an RGD containing protein that is highly expressed in osteoblasts and osteocytes. J Biol Chem 275:3617236180. Qin C, Brunn JC, Jones J, George A, Ramachandran A, Gorski JP, Butler WT. 2001. A comparative study of sialic acidrich proteins in rat bone and dentin. Eur J Oral Sci 109:133 141. Qin C, Brunn JC, Cadena E, Ridall A, Butler WT. 2003. Dentin sialoprotein in bone and dentin sialophosphoprotein gene expressed by osteoblasts. Connect Tissue Res 44:179 183. Qin C, Baba O, Butler WT. 2004. Post-translational modications of sibling proteins and their roles in osteogenesis and dentinogenesis. Crit Rev Oral Biol Med 4:126136.

American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

2546

MACDOUGALL ET AL.

Rajpar MH, Koch MJ, Davies RM, Mellody KT, Kielty CM, Dixon MJ. 2002. Mutation of the signal peptide region of the bicistronic gene DSPP affects translocation to the endoplasmic reticulum and results in defective dentine biomineralization. Hum Molec Genet 11:25592565. Rowe PS, de Zoysa PS, Dong R, Wang HR, White KE, Econs MJ, Oude CL. 2001. MEPE, a new gene expressed in bone marrow and tumors causing osteomalacia. Genomics 67:5468. Senger DR, Wirth DF, Hynes RO. 1979. Transformed mammalian cells secrete specic proteins and phosphoproteins. Cell 16:885893. Shields ED, Bixler D, El-Kafrawy AM. 1973. A proposed classication for heritable human dentine defect with a description of a new entity. Arch Oral Biol 18:543553. Sodek J, Ganss B, McKee MD. 2000. Osteopontin. Crit Rev Oral Biol Med 11:279303. Somerman MJ, Shroff B, Foster B, Foster RA, Butler WT, Sauk JJ. 1992. Mineral-associated adhesion proteins are linked to root formation. Proc Finn Dent Soc 88:451461. Sreenath T, Thyagarajan T, Hall B, Longenecker G, DSouza R, Hong S, Wright JT, MacDougall M, Sauk J, Kulkarni AB. 2003. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III. J Biol Chem 278:2487424880. Thesleff I. 2003. Epithelial-mesenchymal signalling regulating tooth morphogenesis. J Cell Sci 1:16471648.

Veis A. 1993. Mineral-matrix interactions in bone and dentin. J Bone Miner Res 2:S493S497. Waltimo J. 1996. Developmental Defects of Human dentin matrix. PhD Thesis. University of Helsinski, Finland. Witkop CJ. Hereditary defects in enamel and dentin. 1957. Acta Genet 7:236239. Witkop CJ Jr. 1975. Hereditary defects of dentin. Dent Clin N Am 19:2545. Witkop CJ Jr, Rao SR. 1971. Inherited defects in tooth structure. Birth Defects Orig Art Ser VII:153184. Witkop CJ Jr, MacLean CJ, Schmidt PJ, Henry JL. 1966. Medical and dental ndings in the Brandywine isolate. Ala J Med Sci 3:382 403. Xiao S, Yu C, Chou X, Yuan W, Wang Y, Bu L, Fu G, Qian M, Yang J, Shi Y, Hu L, Han B, Wang Z, Huang W, Liu J, Chen Z, Zhao G, Kong X. 2001. Dentinogenesis imperfecta 1 with or without progressive hearing loss is associated with distinct mutations in DSPP. Nature Genet 27:345. Ye L, MacDougall M, Zhang S, Xie Y, Zhang J, Li Z, Lu Y, Mishina Y, Feng JQ. 2004. Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development. J Biol Chem 279:1914119148. Zhang X, Zhao J, Li C, Gao S, Qiu C, Liu P, Wu G, Qiang B, Lo WHY, Shen Y. 2001. DSPP mutation in dentinogenesis imperfecta Shields type II. Nature Genet 27:151152.