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Hereditary dentine diseases resulting from mutations in DSPP gene
journal of dentistry 40 (2012) 542–548
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Review
Hereditary dentine diseases resulting from mutations in DSPP gene§ Izabela Maciejewska *, Ewa Chomik Department of Dental Prosthodontics, Medical University of Gdansk, 18 E. Orzeszkowej St., 80-208 Gdansk, Poland
article info
abstract
Article history:
Objectives: This review groups the newest results of molecular analyses of DSPP gene for
Received 6 September 2011
patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and
Received in revised form
tries to link the phenotypes with specific mutations in the DSPP gene.
4 April 2012
Data: The review includes biochemical data introducing a specificity of DSPP protein which
Accepted 5 April 2012
justifies it as a critical factor for dentine mineralization and maturation. The majority of the review analyzes mutations in the DSPP gene which result in phenotypes of dentinogenesis imperfecta types II or/and III or dentine dysplasia.
Keywords:
Sources: An electronic search was conducted in the databases of Pub Med and supplemented
DSPP
by manual study of relevant references.
Dentinogenesis imperfecta type II/III
Study selection: 52 out of 108 references were finally selected for the review based on the
Dentine dysplasia
novelty and/or originality of data. Conclusion: Hereditary dentine disorders dentinogenesis imperfecta type II/III and dentine dysplasia are currently proposed to be one disease with distinct clinical manifestations reflecting various mutations in the same DSPP gene. For years both disorders were linked exclusively to mutations in the DSP code but a growing number of papers describe mutations which manifest a similar phenotype but are localized in the strongly repetitive sequence of the 30 terminus of the DSPP which codes DPP protein. Our search suggests that the localization of mutation in the sequence of the DSPP gene might result in a different phenotype due to the diverse cellular fate of the mutated protein. Thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is still required. # 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Dentinogenesis is a strictly controlled process in which an extra cellular matrix (ECM) is secreted and subsequently mineralized. These events are under close cellular control by the formative cells, odontoblasts, and any defect of these cells can lead to aberrations of dentine formation with consequent §
effects on tissue function. Mineral represents up to 70% of the mature dentine while the organic phase accounts for 20% and is primarily composed of type I collagen.1,2 About 10% of the organic phase of dentine is composed of various noncollagenous proteins with DPP, DSP and DGP (dentine phosphoprotein, dentine sialoprotein, dentine glycoprotein, respectively) being characteristic components of dentine. These three ECM molecules originate from a native chimeric
Supported by grant: N N403 130040 to IM. * Corresponding author. Tel.: +48 58 349 2159; fax: +48 58 349 2150. E-mail address: [email protected] (I. Maciejewska). 0300-5712/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2012.04.004
journal of dentistry 40 (2012) 542–548
protein – DSPP (dentine sialophosphoprotein), which is not found extracellularly due to intracellular post-translational processing. In humans, DSPP consists of about 1300 amino acids (aa) and is predominantly expressed in teeth (dentine), although its presence in bone and several soft tissues has been detected at much lower3 concentrations. Immediately after the translation of full-length DSPP, proteases from the astacin family cleave DSPP into 3 separate daughter proteins of different properties.4 DSP originates from the N-terminus of DSPP (aa16-374) and is both N- and O-linked glycosylated exclusively with chondroitin 6-sulphate glycan5 in rat and mostly with chondroitin 4-sulphate glycan in bovine.6 The scrutinized analysis of the spatial conformation of DSP showed many differences between species.6–8 Similarly, the differences in the molecular weight of DSP have been reported.9,10 These discrepancies possibly result from the fact that in the ECM of dentine, DSP coexists in two forms: the core protein called DSP and the proteoglycan form called DSP-PG or DPG.6,7,10 Additionally, DSP might form the covalently linked dimmers, which were reported in the cases of both pig and bovine.6,7 DSP’s role in dentine mineralization has not been elucidated yet. However, it is believed that it mediates a very early phase of mineral formation.11,12 Also, the existence of the proteoglycan form of DSP exposed involvement of GAG chains in the process of the transition of predentine into dentine.13 The proteoglycan form of the porcine DGP spans amino acids 375–462, has 4 phosphorylated serine residues and 1 glycosylated asparagine.14 The N-terminus of DPP begins with an AspAspProAsn sequence and extends to the C-terminus for an average 500 aa (in pigs).5,15 The length of the DPP differs between individuals and is believed to represent a polymorphism,16 although this does not appear to affect the DPP’s putative function.17 Due to the large number of Asp, Glu and Ser residues, DPP is highly hydrophilic and one of the body’s most acidic proteins. After post-translational phosphorylation of the Ser residues, mainly localized at the 30 terminus of the DPP, this protein contains an average of 200 phosphates per molecule, which are gathered in the repeating sequences of Asp-Pse-Pse. The analytical study of the spatial conformation of these repeats showed that they produce a ribbon-like, trans-extended chain structure, with the repetitive arrays of the phosphate and carboxylate groups, on both edges of the polypeptide chain.18 This specific spatial arrangement works efficiently for calcium binding and bridging. Thus the strongly anionic nature and spatial conformation of the DPP has contributed to its implication in crystal nucleation and mineral formation.19,20 Nascent and posttranslationally modified DPP migrates immediately to the mineralization front following secretion, where it is incorporated into the framework of the collagenous fibrils and stimulates mineral formation.21 However, the in vitro studies showed that DPP’s role in dentine mineralization is concentration dependent.22,23 Thus DPP stimulates mineralization at low concentration whereas it inhibits it at high concentration.22,23 This inhibitory function of DPP can possibly protect dentine against being over mineralized. Molecular analysis of the human genome has localized the DSPP gene to chromosome 4q22.1. DSPP consists of 5 exons
543
and 4 introns, spanning a total of 8343 base pairs.19 The first exon is non-coding whereas the second codes mainly the sequence of a signal peptide. The DSP sequence is coded by the third and fourth exons and the very short 50 region of exon five. The entire sequence of DPP is coded on exon five.24,25 The sequence of human DPP is highly repetitive and contains an average of 200 tandem copies of 9-bp that encode Ser-Ser-Asp repeats.26 Moreover, recent data has shown that the Cterminus of human DPP sequence displays an allelic polymorphism, which results in variability in the number of Ser-SerAsp repeats in healthy individuals.26,27 The aim of this paper is to assemble the newest results of molecular analyses of the DSPP gene for patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and try to link the phenotypes with specific mutations in the DSPP gene.
2.
Materials and methods
2.1.
Data
This review was written to evaluate the most significant and recent data which emphasize the molecular analysis of mutations in the DSPP gene resulting in phenotypes of dentinogenesis imperfecta type II/III (DGI-II/III) or dentine dysplasia (DD). Only the mutations which result in the phenotype of dentinogenesis imperfecta type II/III or dentine dysplasia were described in the manuscript. In the authors opinion it is critical to query why the same mutation in the DSPP gene results in the phenotype of dentinogenesis imperfecta type II in some individuals, whereas others suffer from dentine dysplasia. Answers to this query can significantly extend our understanding of whether and how a genetic background modifies the final phenotype. The authors took up the challenge of finding correlations (if any) between both the type and localization of the mutation and the specific symptoms presented in clinical evaluation.
2.2.
Sources
An electronic search of Pub Med databases (up to November 2011) included the following subjects in various combinations: #1 DSPP; #2 human; #3 teeth; tooth; dental; #4 dentine; #5 dentinogenesis imperfecta type II/III; #6 dentine dysplasia; #7 hereditary disorders; #8 mice; #9 English The database was searched to include only manuscripts in English. Polish literature was examined manually.
2.3.
Selection criteria
Since both dentinogenesis imperfecta type II/III and dentine dysplasia are very rare hereditary dentine disorders, our preliminary search involved 108 relevant references. The inclusion criteria were manuscripts that evaluated: 1. Composition, formation and structure of dentine. 2. Biochemical data introducing a specificity of DSPP protein. 3. The phenotype of dentinogenesis imperfecta type II/III (DGI-II/ III) and dentine dysplasia (DD).
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journal of dentistry 40 (2012) 542–548
Electronic search of PubMed database utilizing keywords Manual search of Polish references (total 108 manuscripts)
56 manuscripts excluded based on the exclusion criteria
Composition, formation, and structure of dentine
Biochemical data introducing a specificity of DSPP protein
The phenotype of DGI-II/III and DD.
The molecular analysis of mutations within DSPP gene resulting in phenotypes of DGI-II/III and DD
Screening of titles and abstracts (52 manuscripts selected and accepted)
Fig. 1 – Selection criteria for the chosen references.
4. The molecular analysis of mutations within the DSPP gene resulting in phenotypes of DGI-II/III and DD. Under the criteria of novelty and originality, data presented in 52 of them was classified for further evaluation. Letters to the editor, historical reviews, abstracts, posters, chapters in textbooks and unpublished articles were not sought (Fig. 1).
3.
Results and discussion
To date, solely mutations in DSPP have been linked to the isolated hereditary disorders of dentine formation, which were originally categorized as dentinogenesis imperfecta and dentine dysplasia.28 Dentinogenesis imperfecta type II (OMIM 125490), also called opalescent dentine, is reported to have an incidence ranging from 1 in 6000 to 1 in 8000 of live births29; however, the epidemiological data is very limited. Commonly described clinical symptoms include yellow, amber brown or bluish grey discolouration and substantial translucency of the teeth. Histologically, the dentine of these teeth shows a limited number or complete lack of dentine tubules with significantly reduced and defective dentine mineralization. Due to the softness of the underlying dentine, enamel tends to chip off leading to the rapidly progressive attrition of the teeth. Radiologically, teeth show bulbous crowns with cervical constrictions at the root junction. Roots are smaller and narrower compared to healthy teeth. Pulp chambers and root canals are usually obliterated.12,30 The same features clinically diagnosed in the deciduous teeth are classified as dentine dysplasia type II (DD-II)(OMIM 125420). The permanent teeth in patients with dentine dysplasia type II are much less affected; however, radiologically these teeth show thistle-tube pulp chambers with multiple pulp stones.12,16,31 To date, the incidence of dentinogenesis imperfecta type III (OMIM 125500) has been associated with descendents of an isolated tri-racial sub-population known as the ‘‘Brandywine
isolate’’. This population was originally located in Southern Maryland, USA (in the Brandywine river valley) and the incidence of DGI-III has been estimated at 1 in 15 live births in that population.12 Despite close clinical similarities to DGI-II, a significant difference is seen radiographically with the presence of so-called ‘‘shell teeth’’ with enlarged, poorly mineralized pulp chambers and widened root canals.32,33 An animal study confirmed that DSPP knock-out mice show a phenotype that resembles human dentinogenesis imperfecta type III.34 It is important to emphasize that the initial classification of dentinogenesis imperfecta and dentine dysplasia has been based on the clinical and radiologic symptoms without substantial knowledge of the molecular pathogenesis. Thus the challenge of current research is to establish the correlation between particular genetic mutations in the sequence of the DSPP gene and related clinical features manifested in the phenotype. To date, more than 30 mutations in the DSPP gene have been reported,17,35,36 although some of them do not result in the phenotype of dentinogenesis imperfecta type II or dentine dysplasia. Due to ‘‘degeneracy’’ of codons, the mutational change of the third nucleotide in a codon (except ATG and TGG) often remains silent. The same relates to the insertion and/or deletion of three consequent nucleotides (entire codon/ s) which does not cause a frameshift.17 According to the DSPP sequence, mutations have been arbitrarily divided into 3 groups; those that appear in the coding sequence of: (1) the signal peptide, (2) DSP or (3) DPP. It is interesting that in the coding sequences of the signal peptide and/or DSP code, there have been detected mostly the nonsense and/or missense mutations, while the phenotype related mutations that appear in the DPP coding sequence are generally deletions or insertions resulting in a frameshift. Mutations in the sequence of the signal peptide of DSPP have been described in two papers20,37 and both were of a missensenature (Table 1). The first one was reported in codon 6 (T>G) and resulted in the substitution of the hydrophobic Tyr for the hydrophilic Asp at the 6th codon of the hydrophobic
545
journal of dentistry 40 (2012) 542–548
Table 1 – Mutations within the signal peptide of DSPP. Races
Mutation class
Caucasian/German Central American
Missense Missense
cDNA
Amino acid
c.16T>G c.15C>T
Pro17Thr Ala15Val
Predicted protein
Sequences
p.Y6D p.A15V
TAT>GAT GCC>GTC
core of the signal peptide domain.20 This mutation reflected the phenotype of dentine dysplasia II presented by all affected members of the family. In contrast, the transition of C>T in the last 15th codon of the DSPP signal peptide caused the substitution of Ala with Val and resulted in the clinically diagnosed dentinogenesis imperfecta type II.37 It has been proposed that the mutations in the signal peptide of DSPP lead to impaired or no translocation of the protein into the endoplasmic reticulum (ER).20 Subsequently, the protein does not undergo the usual intense post-translational modifications and is degraded in the cellular cytosol or becomes immobilized in the membrane of the ER, reducing its availability and function. Consequently, this reduces the amount of both DSP and DPP secreted into the extracellular matrix, which impacts on the entire process of dentine deposition and mineralization. A similar explanation for the severe phenotype of dentinogenesis imperfecta type II or III in members of a few families has also been proposed32,35,36,38–41 (Table 2). The authors described the transversion of c.49 (C>A or C>T) which resulted in the substitution of p.17 Pro into Thror Ser, respectively39,41,42 as well as the transversion of c.52 (G>T) that caused the substitution of p.18Val into Phe.38,39,43,44
Exon 2 2
Diagnosis
References
DD-II DGI-II
20 37
Since the C-terminus of the signal peptide and the first 3 amino acids (especially Pro at the 2nd and Val at 3rd positions, respectively) of the mature protein contain the signal peptidase cleavage site, it is conceivable that a missense mutation in the first three amino acids of the mature DSPP leads to errors in signal peptide processing; however, such correlations between specific point mutations and the observed phenotype still have to be demonstrated. DSPP mutations that appear in the DSP code and those that appear in the first three amino acids usually manifest a phenotype of dentinogenesis imperfecta type II37–39,43,45 (Table 2). Generally, discolouration of both deciduous and permanent teeth is observed with accompanying cuspal attrition and pulp chamber obliteration.47 In the 50 sequence of DSPP that encodes DSP, a transition was detected particularly in the c.133 (C>T)38,47 of exon 3 and resulted in the nonsense p.Gln 45 stop mutation. The nonsense mutations are responsible for the premature termination of transcription that conceivably results in incomplete translation and truncated DSPP formation; thus only DSP is formed. The phenotype which resulted from the nonsense mutation c.133 (C>T) observed in Song’s study38 revealed the deposition of abnormal dentine with a
Table 2 – Mutations in the coding sequence of DSP, including changes within the conserved Ile–Pro–Val domain and exon 3 skipping. Races
Mutation class
cDNA
Amino acid
Predicted protein
Sequences
Exon/ intron
Diagnosis
References
Brandywine triracial isolate/Chinese Chinese
Missense
c.49C>T
Pro17Ser
p.P17S
CCA>TCA
Exon 2
DGI-II
41,42
Missense
c.49C>A
Pro17Thr
p.P17T
CCA>ACA
Exon 2
39
Chinese
Missense
c.52G>T
Val18Phe
p.V18F
GTT>TTT
Exon 3
Caucasian Finnish Korean/ Chinese Korean
Missense Missense Missense
c.52G>T g.1197G>T c.52G>T
Val18Phe Val18Phe Val18Phe
p. V18F p.V18F p.V18F
GTT>TTT GTT>TTT GTT>TTT
Exon 3 Exon 3 Exon 3
DGI-II with hearing loss DGI-II with hearing loss DGI-II DGI-II DGI-III
Missense
Val18Asp
p.V18D
GTC>GAC
Exon 3
DGI-II
36,40
Japanese
Missense
Val18Asp
p.V18D
GTC>GAC
Exon 3
DGI-II
35
Chinese Chinese Sweden Caucasian Korean
Nonsense Nonsense Missense Missense Splicing site mutation
Gln45 STOP Gln45 STOP Arg68Trp Arg68Trp –
p.Q45X p.Q45X p.R68W p.R68W p.V18_Q45del
CAG>TAG CAG>TAG AGG>TGG AGG>TGG CAG>GAG
Exon 3 Exon 3 Exon 4 Exon 4 Intron 2
DGI-II DGI-II DGI-II DGI-II DGI-II
38
Caucasian/ Finnish Mongolian Chinese
Splicing site mutation
c.53T>A g.1198T>A c.53T>A g.1192T>A c.133C>T c.3658C>T c.68A>T c.202A>T g.1188C>G, IVS23C>G g.1194C>A
–
p.V18_Q45del
CAG>AAG
Intron 2
DGI-II
44
Splicing site mutation Splicing site mutation, skipping of exon 3 Splicing site mutation, skipping of exon 3
IVS3+3A>G c.135+ 1 G>A c.135+ 1 G>T
– –
– p.V18_Q45del
GTAT>GTGT TACAGg/a
Intron 3 Intron 3
DGI-II DGI-II
46
–
p.V18_Q45del
TACAGg/t
Intron 3
DGI-II
26
Caucasian
39
43 44 38,43
47 37 44 48
39
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journal of dentistry 40 (2012) 542–548
reduced number and orientation of dentinal tubules and the presence of dense, amorphous masses inside the dentine tubules. With polymorphism analysis, Xiao et al. described another mutation occurring in the coding sequence of DSP, which revealed a G>A transition (c.135) in the donor splice site of intron 3 possibly resulting in the skipping of the entire exon 3.39 The hypothesis of the skipping of exon 3 was also discussed by McKnight et al.,26 who described the transversion of G>T in the codon 135 and DNA sequence analysis led to their proposal that the mutation is responsible for a loss of function change in the functional acceptor site at the 50 side of the exon 4 splicing sequence, with the 50 bp long sequence void of an alternative strong splice acceptor site. Both those and others mutations44,46–48 that resulted in the skipping of the exon 3 manifested the severe phenotype of dentinogenesis imperfecta type II with progressive attrition and discolouration of teeth and obliteration of pulp chambers; however, there were no accompanying reports of progressive hearing loss.26,39,46 It is difficult to speculate whether the mutations (but not nonsense mutation) that occur in the DSP sequence of the DSPP gene result exclusively in the disruption of DSP formation or if they influence the DPP protein as well. Research on biomineralization suggests that DSP mediates early dentine formation and the different pools of proteoglycans, isolated from different fractions of dentine (predentine, dentine-predentine interface, dentine) mediate the transition of predentine into dentine,13,49 including the timing of fibrillogenesis along with fibrils alignment and aggregation. Thus the phenotype resulting from mutations in the DSP sequence might support the hypothesis that the role of DSP and/or DSP-PG in dentine formation is irreplaceable and cannot be balanced by any other protein of the extracellular matrix of dentine. Recently, considerable research indicates that the greatest diversity in phenotypes reflecting disrupted dentine formation is manifested by mutations that appear in exon 5 of the DSPP gene which encodes entire DPP26 (Table 3). To date, all mutations discovered in exon 5 are deletions and/or insertions with an accompanying frameshift.17,26,32,44,50,51 The first comprehensive study of the DSPP sequence, which included the repetitive domain of over 200 tandem copies of 9 bp repeats, was described by McKnight et al.26,51 The authors reported five different mutations which resulted in the reading
frameshift that consequently led to a change in the repetitive sequences of three hydrophilic and phosphorylated aminoacids (Ser-Ser-Asp) into a long sequence that encoded hydrophobic amino acids like Val, Ala, Ile. DNA sequencing showed either one or four nucleotide deletions that were localized in the 30 terminal region of the DSPP gene (c.1870del TCAG; c.1918del TCAG; c.2272del A; c.2525del G; c.3141del C, respectively). The results of McKnight’s study showed that hundreds of changed amino acids at the 30 terminal fragment of DPP resulted in the phenotypes of both dentine dysplasia and/or dentinogenesis imperfecta type II. Surprisingly, the authors suggested that the longer sequence change (600 codons) resulted in the phenotype of dentine dysplasia, whereas a change in the shorter hydrophobic sequence (<500 codons) was manifested by the permanent teeth injury classified as dentinogenesis imperfecta type II. Although they carried out detailed studies, the authors were not able to define why the specific deletion caused the phenotype of dentine dysplasia whereas others resulted in dentinogenesis imperfecta type II and speculated that this might be connected with gene polymorphism and hyplotypes formation. Thus the final phenotype reflected the activity of a dominant allele, which might be mutated or not.26,51 The great diversity in haplotypes of the DSPP sequence and difficulty in sequencing the highly repetitive 30 terminal fragment of the gene have made it difficult to conduct a full analysis of exon 5. It is only recently that a growing number of studies have suggested that the majority of cases of hereditary dentine disorders might result from mutations causing the reading frameshift in the 30 terminal fragment of exon 5. A recent comprehensive study, based on kindred from 12 unrelated families, has linked the specific phenotypes to the type of mutation.17 It was shown that short span deletions (1–4 nt) which localized closer to the 50 terminal fragment of the DPP code were manifested by milder tooth discolouration and smaller changes in pulp chamber anatomy than deletions that appeared in the most repetitive 30 terminal fragment of DPP. Additionally, it was shown that the more codons that were deleted, the more severe the phenotype was. This phenotype manifested dark discolouration of both dentition, complete chamber obliteration, and severe teeth attrition, eventually followed by frequent periapical infections. The Nieminen et al.’s study clearly demonstrated that only frameshift
Table 3 – Mutations within DPP code. Races/country
Mutation class
cDNA
Brandywine Isolate/USA
Insertion/deletion
Caucasian Caucasian Greek Caucasian/Northern European Caucasian/Northern European Caucasian/Finnish Caucasian/Finnish Korean Korean Caucasian/Finnish Caucasian/Finnish Not reported
Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift
c.3599_3634 del 36bp c.3715_3716 ins 18bp c.1870delTCAG c.1918delTCAG c.1918delTCAG c.2272delA c.2525delG c.2063delA c.3582del 10bp c.2688delT c.3560delG g.3599del34bp g.3715ins2bp c.3141delC
Predicted protein
Exon
Diagnosis
References
–
5
DGI-III
32
p.S624TfsX687 p.S640TfsX671 p.S640TfsX671 p.S758AfsX554 p.S842TfsX471 – – – – – – –
5 5 5 5 5 5 5 5 5 5 5 5
DD-II DD-II DD-II DGI-II DGI-II DD-II DGI-III DGI-II DGI-II DGI-III DGI-III DD-II
26 26 17 26 26 17 17 50 50 44 44 51
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mutations led to the phenotypes of dentinogenesis imperfecta type II or dentine dysplasia, whereas in frame mutations did not cause observable clinical changes.17
4.
Conclusions
Even though the sophisticated methods used currently for molecular analysis of the DSPP gene precisely define types and localizations of DSPP mutations, the direct correlation between the specific mutation and manifested clinical symptoms is not possible yet. Possibly, there are many other contributing factors together with environmental ones52 which, acting in concert with genetics, influence the final phenotype observed in the patients diagnosed with dentinogenesis imperfecta type II or dentine dysplasia. Clinical diagnosis of dentinogenesis imperfecta type II and dentine dysplasia includes recognition of a cluster of symptoms that accompany this hereditary dentine disorder(s), although individual cases differ one from another in regard to teeth discolouration, cervical region constriction, presence of pulp stones, severity of teeth attrition or coexistence of periapical inflammation. Thus detailed progressive clinical evaluations of probants and their multi-generation families seem critical. The histological study of mutated teeth, both deciduous and permanent, could bring more data as well. It is not clear yet whether the mutated DSPP molecule is processed in ER and subsequent organelles prior to final release into the extracellular matrix of dentine or if a change in the chemical properties of DSPP results in aggregation of unprocessed DSPP in the cytosol of odontoblasts. It might be speculated that the most frequent mutations, which appear in the code for the signal peptide and/or three first amino acids of the coding sequence of DSPP, impact on the transport and processing of the mutated protein, leading to it becoming trapped and blocking cellular events in the rough ER. This might eventually affect translation of DSPP and also other proteins resulting in disrupted dentine formation. However, the reading frameshift mutations of the DPP code change the properties of DPP, which may disrupt the entire process of dentine maturation, eventually resulting in the severe phenotype of dentinogenesis imperfecta type II and/or dentine dysplasia. There is thus considerable scope for further research at the cellular level to determine the mechanistic effects of DSPP mutations. So far researchers mostly speculate about the potential protein formation, predicting the final protein sequence from the analysis of the sequence of mutated DNA, although the most specific data would come from the direct co-evaluation of both the mutated gene and protein. Such a detailed analysis should precisely answer the query about what ‘‘protein’’ is formed from the specifically mutated gene. The use of DSPP gene sequencing, especially for the repetitive sequence of the DPP code, is important in the clinical diagnosis of patients with dentinogenesis imperfecta type II/III and dentine dysplasia, but our understanding of the cellular fate of mutated DSPP is still unclear. Such knowledge may help to underpin future molecular therapies for these patients and thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is required.
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Acknowledgement The authors acknowledge the helpful advice of Dr. AJ Smith and Mr. Michael Liley.
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18. George A, Bannon L, Sabsay B, Dillon JW, Malone J, Veis A, et al. The carboxyl-terminal 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. The Journal of Biological Chemistry 1996;271:32869–73. 19. Mac Dougall M, Simmons D, Luan X, Gu TT, Du Pont BR. Assignment of dentin sialophosphoprotein (DSPP) to the critical DGI2 locus on human chromosome 4 band q21.3 by in situ hybridization. Cytogenetics and Cell Genetics 1997;79:121–2. 20. Rajpar MH, Koch MJ, Davies RM, Mellody KT, Kielty CM, Dixon MJ. Mutation of the signal peptide region of the bicistronic gene DSPP affects translocation to the endoplasmic reticulum and results in defective dentine biomineralization. Human Molecular Genetics 2002; 11:2559–65. 21. Butler WT, Ritchie HH, Bronckers AL. Extracellular matrix proteins of dentine. Ciba Foundation Symposium 1997;205:107–15. 22. Boskey A, Spevak L, Tan M, Doty SB, Butler WT. Dentin sialoprotein (DSP) has limited effects on in vitro apatite formation and growth. Calcified Tissue International 2000;67:472–8. 23. Boskey A, Maresca M, Doty S, Sabsay. Veis A. Concentrationdependent effects of dentin phosphophoryn in the regulation of in vitro hydroxyapatite formation and growth. Bone Mineral 1990;11:55–65. 24. Fedarko NS, Jain A, Karadag A, Fisher LW. Three small integrin binding ligand N-linked glycoproteins (SIBLINGs) bind and activate specific matrix metalloproteinases. FASEB Journal 2004;18:734–6. 25. Fisher LW, Fedarko NS. Six genes expressed in bones and teeth encode the current members of the SIBLING family of proteins. Connective Tissue Research 2003;44:33–40. 26. McKnight DA, Hart PS, Hart TC, Hartsfield JK, Wilson A, Wright JT, et al. A comprehensive analysis of normal variation and disease-causing mutations in the human DSPP gene. Human Mutation 2008;29:1392–404. 27. Beattie ML, Kim JW, Gong SG, Murdoch-Kinch CA, Simmer JP, Hu JCC. Phenotypic variation in dentinogenesis imperfecta/dentin dysplasia linked to 4q21. Journal of Dental Research 2006;85:329–33. 28. Shields ED, Bixler D, el-Kafrawy AM. A proposed classification for heritable human dentine defects with a description of a new entity. Archives of Oral Biology 1973;18:543–53. 29. Witkop CJ, MacLean PJ, Schmidt JL. Medical and dental findings in the Brandywine isolate. Alabama Journal of Medical Sciences 1966;3:382–403. 30. Acevedo AC, Santos LJ, Paula LM, Dong J, Mac Dougall M. Phenotype characterization and DSPP mutational analysis of three Brazilian dentinogenesis imperfecta type II families. Cells Tissues Organs 2009;189:230–6. 31. Barron MJ, McDonnell ST, Mackie I, Dixon MJ. Hereditary dentine disorders: dentinogenesis imperfecta and dentine dysplasia. Orphanet Journal of Rare Diseases 2008;3:31. 32. Dong J, Gu T, Jeffords L, Mac Dougall M. Dentin phosphoprotein compound mutation in dentin sialophosphoprotein causes dentinogenesis imperfecta type III. American Journal of Medical Genetics A 2005;132A:305–9. 33. Bixler D, Conneally PM, Christen G. Dentinogenesis imperfecta: genetic variations in a six-generation family. Journal of Dental Research 1969;48:1196–9. 34. Sreenath T, Thyagarajan T, Hall B, Longenecker G, D’Souza R, Hong S, et al. Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human
35.
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dentinogenesis imperfecta type III. Journal of Biological Chemistry 2003;278:24874–80. Kida M, Tsutsumi T, Shindoh M, Ikeda H, Ariga T. De novo mutation in the DSPP gene associated with dentinogenesis imperfecta type II in a Japanese family. European Journal of Oral Sciences 2009;117:691–4. Lee SK, Lee KE, Hwang YH, Kida M, Tsutsumi T, Ariga T, et al. Identification of the DSPP mutation in a new kindred and phenotype-genotype correlation. Oral Diseases 2011; 17:314–9. Malmgren B, Lindskog S, Elgadi A, Norgren S. Clinical, histopathologic, and genetic investigation in two large families with dentinogenesis imperfecta type II. Human Genetics 2004;114:491–8. Song Y, Wang C, Peng B, Ye X, Zhao G, Fan M, et al. Phenotypes and genotypes in 2 DGI families with different DSPP mutations. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 2006;102:360–74. Xiao S, Yu C, Chou X, Yuan W, Wang Y, Bu L, et al. Dentinogenesis imperfecta 1 with or without progressive hearing loss is associated with distinct mutations in DSPP. Nature Genetics 2001;27:201–4. Lee SK, Lee KE, Jeon D, Lee G, Lee H, Shin CU, et al. A novel mutation in the DSPP gene associated with dentinogenesis imperfecta type II. Journal of Dental Research 2009;88:51–5. Hart PS, Hart TC. Disorders of human dentin. Cells Tissues Organs 2007;186:70–7. Zhang X, Chen L, Liu J, Zhao Z, Qu E, Wang X, et al. A novel DSPP mutation is associated with type II dentinogenesis imperfecta in a Chinese family. BMC Medical Genetics 2007;8:52–7. Kim JW, Hu JCC, Lee J, Moon SK, Kim YJ, Jang KT, et al. Mutational hot spot in the DSPP gene causing dentinogenesis imperfecta type II. Human Genetics 2005;116:186–91. Holappa H, Nieminen P, Tolva P, Lukinmaa PL, Alaluusua S. Splicing site mutations in dentin sialophosphoprotein causing dentinogenesis imperfecta type II. European Journal of Oral Sciences 2006;114:381–4. Lee SK, Hu JC, Lee KE, Simmer JP, Kim JW. A dentin sialophosphoprotein mutation that partially disrupts a splice acceptor site causes type II dentin dysplasia. Journal of Endodontics 2008;34:1470–3. Bai H, Agula H, Wu Q, Zhou W, Sun Y, Qi Yue. et al. A novel DSPP mutation causes dentinogenesis imperfecta type II in a large Mongolian family. BMC Medical Genetics 2010;11:23. Zhang X, Zhao J, Li C, Gao S, Qiu C, Liu P, et al. DSPP mutation in dentinogenesis imperfecta Shields type II. Natural Genetics 2001;27:151–2. Kim JW, Nam SH, Jang KT, Lee SH, Kim CC, Hahn SH, et al. A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II. Human Genetics 2004;115:248–54. Waddington RJ, Hall RC, Embery G, Lloyd DM. Changing profiles of proteoglycans in the transition of predentine to dentine. Matrix Biology 2003;22:153–61. Lee KE, Kang HY, Lee SK, Yoo SH, Lee JC, Hwang YH, et al. Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II. Clinical Genetics 2011;79:378–84. McKnight DA, Simmer JP, Hart PS, Hart TC, Fisher LW. Overlapping DSPP mutations cause dentin dysplasia and dentinogenesis imperfecta. Journal of Dental Research 2008;87:1108–11. Atar M, Korperich EJ. Systemic disorders and their influence on the development of dental hard tissues. A literature review. Journal of Dentistry 2010;38:296–306.
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Review
Hereditary dentine diseases resulting from mutations in DSPP gene§ Izabela Maciejewska *, Ewa Chomik Department of Dental Prosthodontics, Medical University of Gdansk, 18 E. Orzeszkowej St., 80-208 Gdansk, Poland
article info
abstract
Article history:
Objectives: This review groups the newest results of molecular analyses of DSPP gene for
Received 6 September 2011
patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and
Received in revised form
tries to link the phenotypes with specific mutations in the DSPP gene.
4 April 2012
Data: The review includes biochemical data introducing a specificity of DSPP protein which
Accepted 5 April 2012
justifies it as a critical factor for dentine mineralization and maturation. The majority of the review analyzes mutations in the DSPP gene which result in phenotypes of dentinogenesis imperfecta types II or/and III or dentine dysplasia.
Keywords:
Sources: An electronic search was conducted in the databases of Pub Med and supplemented
DSPP
by manual study of relevant references.
Dentinogenesis imperfecta type II/III
Study selection: 52 out of 108 references were finally selected for the review based on the
Dentine dysplasia
novelty and/or originality of data. Conclusion: Hereditary dentine disorders dentinogenesis imperfecta type II/III and dentine dysplasia are currently proposed to be one disease with distinct clinical manifestations reflecting various mutations in the same DSPP gene. For years both disorders were linked exclusively to mutations in the DSP code but a growing number of papers describe mutations which manifest a similar phenotype but are localized in the strongly repetitive sequence of the 30 terminus of the DSPP which codes DPP protein. Our search suggests that the localization of mutation in the sequence of the DSPP gene might result in a different phenotype due to the diverse cellular fate of the mutated protein. Thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is still required. # 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Dentinogenesis is a strictly controlled process in which an extra cellular matrix (ECM) is secreted and subsequently mineralized. These events are under close cellular control by the formative cells, odontoblasts, and any defect of these cells can lead to aberrations of dentine formation with consequent §
effects on tissue function. Mineral represents up to 70% of the mature dentine while the organic phase accounts for 20% and is primarily composed of type I collagen.1,2 About 10% of the organic phase of dentine is composed of various noncollagenous proteins with DPP, DSP and DGP (dentine phosphoprotein, dentine sialoprotein, dentine glycoprotein, respectively) being characteristic components of dentine. These three ECM molecules originate from a native chimeric
Supported by grant: N N403 130040 to IM. * Corresponding author. Tel.: +48 58 349 2159; fax: +48 58 349 2150. E-mail address: [email protected] (I. Maciejewska). 0300-5712/$ – see front matter # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdent.2012.04.004
journal of dentistry 40 (2012) 542–548
protein – DSPP (dentine sialophosphoprotein), which is not found extracellularly due to intracellular post-translational processing. In humans, DSPP consists of about 1300 amino acids (aa) and is predominantly expressed in teeth (dentine), although its presence in bone and several soft tissues has been detected at much lower3 concentrations. Immediately after the translation of full-length DSPP, proteases from the astacin family cleave DSPP into 3 separate daughter proteins of different properties.4 DSP originates from the N-terminus of DSPP (aa16-374) and is both N- and O-linked glycosylated exclusively with chondroitin 6-sulphate glycan5 in rat and mostly with chondroitin 4-sulphate glycan in bovine.6 The scrutinized analysis of the spatial conformation of DSP showed many differences between species.6–8 Similarly, the differences in the molecular weight of DSP have been reported.9,10 These discrepancies possibly result from the fact that in the ECM of dentine, DSP coexists in two forms: the core protein called DSP and the proteoglycan form called DSP-PG or DPG.6,7,10 Additionally, DSP might form the covalently linked dimmers, which were reported in the cases of both pig and bovine.6,7 DSP’s role in dentine mineralization has not been elucidated yet. However, it is believed that it mediates a very early phase of mineral formation.11,12 Also, the existence of the proteoglycan form of DSP exposed involvement of GAG chains in the process of the transition of predentine into dentine.13 The proteoglycan form of the porcine DGP spans amino acids 375–462, has 4 phosphorylated serine residues and 1 glycosylated asparagine.14 The N-terminus of DPP begins with an AspAspProAsn sequence and extends to the C-terminus for an average 500 aa (in pigs).5,15 The length of the DPP differs between individuals and is believed to represent a polymorphism,16 although this does not appear to affect the DPP’s putative function.17 Due to the large number of Asp, Glu and Ser residues, DPP is highly hydrophilic and one of the body’s most acidic proteins. After post-translational phosphorylation of the Ser residues, mainly localized at the 30 terminus of the DPP, this protein contains an average of 200 phosphates per molecule, which are gathered in the repeating sequences of Asp-Pse-Pse. The analytical study of the spatial conformation of these repeats showed that they produce a ribbon-like, trans-extended chain structure, with the repetitive arrays of the phosphate and carboxylate groups, on both edges of the polypeptide chain.18 This specific spatial arrangement works efficiently for calcium binding and bridging. Thus the strongly anionic nature and spatial conformation of the DPP has contributed to its implication in crystal nucleation and mineral formation.19,20 Nascent and posttranslationally modified DPP migrates immediately to the mineralization front following secretion, where it is incorporated into the framework of the collagenous fibrils and stimulates mineral formation.21 However, the in vitro studies showed that DPP’s role in dentine mineralization is concentration dependent.22,23 Thus DPP stimulates mineralization at low concentration whereas it inhibits it at high concentration.22,23 This inhibitory function of DPP can possibly protect dentine against being over mineralized. Molecular analysis of the human genome has localized the DSPP gene to chromosome 4q22.1. DSPP consists of 5 exons
543
and 4 introns, spanning a total of 8343 base pairs.19 The first exon is non-coding whereas the second codes mainly the sequence of a signal peptide. The DSP sequence is coded by the third and fourth exons and the very short 50 region of exon five. The entire sequence of DPP is coded on exon five.24,25 The sequence of human DPP is highly repetitive and contains an average of 200 tandem copies of 9-bp that encode Ser-Ser-Asp repeats.26 Moreover, recent data has shown that the Cterminus of human DPP sequence displays an allelic polymorphism, which results in variability in the number of Ser-SerAsp repeats in healthy individuals.26,27 The aim of this paper is to assemble the newest results of molecular analyses of the DSPP gene for patients diagnosed either with dentinogenesis imperfecta type II/III or dentine dysplasia and try to link the phenotypes with specific mutations in the DSPP gene.
2.
Materials and methods
2.1.
Data
This review was written to evaluate the most significant and recent data which emphasize the molecular analysis of mutations in the DSPP gene resulting in phenotypes of dentinogenesis imperfecta type II/III (DGI-II/III) or dentine dysplasia (DD). Only the mutations which result in the phenotype of dentinogenesis imperfecta type II/III or dentine dysplasia were described in the manuscript. In the authors opinion it is critical to query why the same mutation in the DSPP gene results in the phenotype of dentinogenesis imperfecta type II in some individuals, whereas others suffer from dentine dysplasia. Answers to this query can significantly extend our understanding of whether and how a genetic background modifies the final phenotype. The authors took up the challenge of finding correlations (if any) between both the type and localization of the mutation and the specific symptoms presented in clinical evaluation.
2.2.
Sources
An electronic search of Pub Med databases (up to November 2011) included the following subjects in various combinations: #1 DSPP; #2 human; #3 teeth; tooth; dental; #4 dentine; #5 dentinogenesis imperfecta type II/III; #6 dentine dysplasia; #7 hereditary disorders; #8 mice; #9 English The database was searched to include only manuscripts in English. Polish literature was examined manually.
2.3.
Selection criteria
Since both dentinogenesis imperfecta type II/III and dentine dysplasia are very rare hereditary dentine disorders, our preliminary search involved 108 relevant references. The inclusion criteria were manuscripts that evaluated: 1. Composition, formation and structure of dentine. 2. Biochemical data introducing a specificity of DSPP protein. 3. The phenotype of dentinogenesis imperfecta type II/III (DGI-II/ III) and dentine dysplasia (DD).
544
journal of dentistry 40 (2012) 542–548
Electronic search of PubMed database utilizing keywords Manual search of Polish references (total 108 manuscripts)
56 manuscripts excluded based on the exclusion criteria
Composition, formation, and structure of dentine
Biochemical data introducing a specificity of DSPP protein
The phenotype of DGI-II/III and DD.
The molecular analysis of mutations within DSPP gene resulting in phenotypes of DGI-II/III and DD
Screening of titles and abstracts (52 manuscripts selected and accepted)
Fig. 1 – Selection criteria for the chosen references.
4. The molecular analysis of mutations within the DSPP gene resulting in phenotypes of DGI-II/III and DD. Under the criteria of novelty and originality, data presented in 52 of them was classified for further evaluation. Letters to the editor, historical reviews, abstracts, posters, chapters in textbooks and unpublished articles were not sought (Fig. 1).
3.
Results and discussion
To date, solely mutations in DSPP have been linked to the isolated hereditary disorders of dentine formation, which were originally categorized as dentinogenesis imperfecta and dentine dysplasia.28 Dentinogenesis imperfecta type II (OMIM 125490), also called opalescent dentine, is reported to have an incidence ranging from 1 in 6000 to 1 in 8000 of live births29; however, the epidemiological data is very limited. Commonly described clinical symptoms include yellow, amber brown or bluish grey discolouration and substantial translucency of the teeth. Histologically, the dentine of these teeth shows a limited number or complete lack of dentine tubules with significantly reduced and defective dentine mineralization. Due to the softness of the underlying dentine, enamel tends to chip off leading to the rapidly progressive attrition of the teeth. Radiologically, teeth show bulbous crowns with cervical constrictions at the root junction. Roots are smaller and narrower compared to healthy teeth. Pulp chambers and root canals are usually obliterated.12,30 The same features clinically diagnosed in the deciduous teeth are classified as dentine dysplasia type II (DD-II)(OMIM 125420). The permanent teeth in patients with dentine dysplasia type II are much less affected; however, radiologically these teeth show thistle-tube pulp chambers with multiple pulp stones.12,16,31 To date, the incidence of dentinogenesis imperfecta type III (OMIM 125500) has been associated with descendents of an isolated tri-racial sub-population known as the ‘‘Brandywine
isolate’’. This population was originally located in Southern Maryland, USA (in the Brandywine river valley) and the incidence of DGI-III has been estimated at 1 in 15 live births in that population.12 Despite close clinical similarities to DGI-II, a significant difference is seen radiographically with the presence of so-called ‘‘shell teeth’’ with enlarged, poorly mineralized pulp chambers and widened root canals.32,33 An animal study confirmed that DSPP knock-out mice show a phenotype that resembles human dentinogenesis imperfecta type III.34 It is important to emphasize that the initial classification of dentinogenesis imperfecta and dentine dysplasia has been based on the clinical and radiologic symptoms without substantial knowledge of the molecular pathogenesis. Thus the challenge of current research is to establish the correlation between particular genetic mutations in the sequence of the DSPP gene and related clinical features manifested in the phenotype. To date, more than 30 mutations in the DSPP gene have been reported,17,35,36 although some of them do not result in the phenotype of dentinogenesis imperfecta type II or dentine dysplasia. Due to ‘‘degeneracy’’ of codons, the mutational change of the third nucleotide in a codon (except ATG and TGG) often remains silent. The same relates to the insertion and/or deletion of three consequent nucleotides (entire codon/ s) which does not cause a frameshift.17 According to the DSPP sequence, mutations have been arbitrarily divided into 3 groups; those that appear in the coding sequence of: (1) the signal peptide, (2) DSP or (3) DPP. It is interesting that in the coding sequences of the signal peptide and/or DSP code, there have been detected mostly the nonsense and/or missense mutations, while the phenotype related mutations that appear in the DPP coding sequence are generally deletions or insertions resulting in a frameshift. Mutations in the sequence of the signal peptide of DSPP have been described in two papers20,37 and both were of a missensenature (Table 1). The first one was reported in codon 6 (T>G) and resulted in the substitution of the hydrophobic Tyr for the hydrophilic Asp at the 6th codon of the hydrophobic
545
journal of dentistry 40 (2012) 542–548
Table 1 – Mutations within the signal peptide of DSPP. Races
Mutation class
Caucasian/German Central American
Missense Missense
cDNA
Amino acid
c.16T>G c.15C>T
Pro17Thr Ala15Val
Predicted protein
Sequences
p.Y6D p.A15V
TAT>GAT GCC>GTC
core of the signal peptide domain.20 This mutation reflected the phenotype of dentine dysplasia II presented by all affected members of the family. In contrast, the transition of C>T in the last 15th codon of the DSPP signal peptide caused the substitution of Ala with Val and resulted in the clinically diagnosed dentinogenesis imperfecta type II.37 It has been proposed that the mutations in the signal peptide of DSPP lead to impaired or no translocation of the protein into the endoplasmic reticulum (ER).20 Subsequently, the protein does not undergo the usual intense post-translational modifications and is degraded in the cellular cytosol or becomes immobilized in the membrane of the ER, reducing its availability and function. Consequently, this reduces the amount of both DSP and DPP secreted into the extracellular matrix, which impacts on the entire process of dentine deposition and mineralization. A similar explanation for the severe phenotype of dentinogenesis imperfecta type II or III in members of a few families has also been proposed32,35,36,38–41 (Table 2). The authors described the transversion of c.49 (C>A or C>T) which resulted in the substitution of p.17 Pro into Thror Ser, respectively39,41,42 as well as the transversion of c.52 (G>T) that caused the substitution of p.18Val into Phe.38,39,43,44
Exon 2 2
Diagnosis
References
DD-II DGI-II
20 37
Since the C-terminus of the signal peptide and the first 3 amino acids (especially Pro at the 2nd and Val at 3rd positions, respectively) of the mature protein contain the signal peptidase cleavage site, it is conceivable that a missense mutation in the first three amino acids of the mature DSPP leads to errors in signal peptide processing; however, such correlations between specific point mutations and the observed phenotype still have to be demonstrated. DSPP mutations that appear in the DSP code and those that appear in the first three amino acids usually manifest a phenotype of dentinogenesis imperfecta type II37–39,43,45 (Table 2). Generally, discolouration of both deciduous and permanent teeth is observed with accompanying cuspal attrition and pulp chamber obliteration.47 In the 50 sequence of DSPP that encodes DSP, a transition was detected particularly in the c.133 (C>T)38,47 of exon 3 and resulted in the nonsense p.Gln 45 stop mutation. The nonsense mutations are responsible for the premature termination of transcription that conceivably results in incomplete translation and truncated DSPP formation; thus only DSP is formed. The phenotype which resulted from the nonsense mutation c.133 (C>T) observed in Song’s study38 revealed the deposition of abnormal dentine with a
Table 2 – Mutations in the coding sequence of DSP, including changes within the conserved Ile–Pro–Val domain and exon 3 skipping. Races
Mutation class
cDNA
Amino acid
Predicted protein
Sequences
Exon/ intron
Diagnosis
References
Brandywine triracial isolate/Chinese Chinese
Missense
c.49C>T
Pro17Ser
p.P17S
CCA>TCA
Exon 2
DGI-II
41,42
Missense
c.49C>A
Pro17Thr
p.P17T
CCA>ACA
Exon 2
39
Chinese
Missense
c.52G>T
Val18Phe
p.V18F
GTT>TTT
Exon 3
Caucasian Finnish Korean/ Chinese Korean
Missense Missense Missense
c.52G>T g.1197G>T c.52G>T
Val18Phe Val18Phe Val18Phe
p. V18F p.V18F p.V18F
GTT>TTT GTT>TTT GTT>TTT
Exon 3 Exon 3 Exon 3
DGI-II with hearing loss DGI-II with hearing loss DGI-II DGI-II DGI-III
Missense
Val18Asp
p.V18D
GTC>GAC
Exon 3
DGI-II
36,40
Japanese
Missense
Val18Asp
p.V18D
GTC>GAC
Exon 3
DGI-II
35
Chinese Chinese Sweden Caucasian Korean
Nonsense Nonsense Missense Missense Splicing site mutation
Gln45 STOP Gln45 STOP Arg68Trp Arg68Trp –
p.Q45X p.Q45X p.R68W p.R68W p.V18_Q45del
CAG>TAG CAG>TAG AGG>TGG AGG>TGG CAG>GAG
Exon 3 Exon 3 Exon 4 Exon 4 Intron 2
DGI-II DGI-II DGI-II DGI-II DGI-II
38
Caucasian/ Finnish Mongolian Chinese
Splicing site mutation
c.53T>A g.1198T>A c.53T>A g.1192T>A c.133C>T c.3658C>T c.68A>T c.202A>T g.1188C>G, IVS23C>G g.1194C>A
–
p.V18_Q45del
CAG>AAG
Intron 2
DGI-II
44
Splicing site mutation Splicing site mutation, skipping of exon 3 Splicing site mutation, skipping of exon 3
IVS3+3A>G c.135+ 1 G>A c.135+ 1 G>T
– –
– p.V18_Q45del
GTAT>GTGT TACAGg/a
Intron 3 Intron 3
DGI-II DGI-II
46
–
p.V18_Q45del
TACAGg/t
Intron 3
DGI-II
26
Caucasian
39
43 44 38,43
47 37 44 48
39
546
journal of dentistry 40 (2012) 542–548
reduced number and orientation of dentinal tubules and the presence of dense, amorphous masses inside the dentine tubules. With polymorphism analysis, Xiao et al. described another mutation occurring in the coding sequence of DSP, which revealed a G>A transition (c.135) in the donor splice site of intron 3 possibly resulting in the skipping of the entire exon 3.39 The hypothesis of the skipping of exon 3 was also discussed by McKnight et al.,26 who described the transversion of G>T in the codon 135 and DNA sequence analysis led to their proposal that the mutation is responsible for a loss of function change in the functional acceptor site at the 50 side of the exon 4 splicing sequence, with the 50 bp long sequence void of an alternative strong splice acceptor site. Both those and others mutations44,46–48 that resulted in the skipping of the exon 3 manifested the severe phenotype of dentinogenesis imperfecta type II with progressive attrition and discolouration of teeth and obliteration of pulp chambers; however, there were no accompanying reports of progressive hearing loss.26,39,46 It is difficult to speculate whether the mutations (but not nonsense mutation) that occur in the DSP sequence of the DSPP gene result exclusively in the disruption of DSP formation or if they influence the DPP protein as well. Research on biomineralization suggests that DSP mediates early dentine formation and the different pools of proteoglycans, isolated from different fractions of dentine (predentine, dentine-predentine interface, dentine) mediate the transition of predentine into dentine,13,49 including the timing of fibrillogenesis along with fibrils alignment and aggregation. Thus the phenotype resulting from mutations in the DSP sequence might support the hypothesis that the role of DSP and/or DSP-PG in dentine formation is irreplaceable and cannot be balanced by any other protein of the extracellular matrix of dentine. Recently, considerable research indicates that the greatest diversity in phenotypes reflecting disrupted dentine formation is manifested by mutations that appear in exon 5 of the DSPP gene which encodes entire DPP26 (Table 3). To date, all mutations discovered in exon 5 are deletions and/or insertions with an accompanying frameshift.17,26,32,44,50,51 The first comprehensive study of the DSPP sequence, which included the repetitive domain of over 200 tandem copies of 9 bp repeats, was described by McKnight et al.26,51 The authors reported five different mutations which resulted in the reading
frameshift that consequently led to a change in the repetitive sequences of three hydrophilic and phosphorylated aminoacids (Ser-Ser-Asp) into a long sequence that encoded hydrophobic amino acids like Val, Ala, Ile. DNA sequencing showed either one or four nucleotide deletions that were localized in the 30 terminal region of the DSPP gene (c.1870del TCAG; c.1918del TCAG; c.2272del A; c.2525del G; c.3141del C, respectively). The results of McKnight’s study showed that hundreds of changed amino acids at the 30 terminal fragment of DPP resulted in the phenotypes of both dentine dysplasia and/or dentinogenesis imperfecta type II. Surprisingly, the authors suggested that the longer sequence change (600 codons) resulted in the phenotype of dentine dysplasia, whereas a change in the shorter hydrophobic sequence (<500 codons) was manifested by the permanent teeth injury classified as dentinogenesis imperfecta type II. Although they carried out detailed studies, the authors were not able to define why the specific deletion caused the phenotype of dentine dysplasia whereas others resulted in dentinogenesis imperfecta type II and speculated that this might be connected with gene polymorphism and hyplotypes formation. Thus the final phenotype reflected the activity of a dominant allele, which might be mutated or not.26,51 The great diversity in haplotypes of the DSPP sequence and difficulty in sequencing the highly repetitive 30 terminal fragment of the gene have made it difficult to conduct a full analysis of exon 5. It is only recently that a growing number of studies have suggested that the majority of cases of hereditary dentine disorders might result from mutations causing the reading frameshift in the 30 terminal fragment of exon 5. A recent comprehensive study, based on kindred from 12 unrelated families, has linked the specific phenotypes to the type of mutation.17 It was shown that short span deletions (1–4 nt) which localized closer to the 50 terminal fragment of the DPP code were manifested by milder tooth discolouration and smaller changes in pulp chamber anatomy than deletions that appeared in the most repetitive 30 terminal fragment of DPP. Additionally, it was shown that the more codons that were deleted, the more severe the phenotype was. This phenotype manifested dark discolouration of both dentition, complete chamber obliteration, and severe teeth attrition, eventually followed by frequent periapical infections. The Nieminen et al.’s study clearly demonstrated that only frameshift
Table 3 – Mutations within DPP code. Races/country
Mutation class
cDNA
Brandywine Isolate/USA
Insertion/deletion
Caucasian Caucasian Greek Caucasian/Northern European Caucasian/Northern European Caucasian/Finnish Caucasian/Finnish Korean Korean Caucasian/Finnish Caucasian/Finnish Not reported
Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift Frameshift
c.3599_3634 del 36bp c.3715_3716 ins 18bp c.1870delTCAG c.1918delTCAG c.1918delTCAG c.2272delA c.2525delG c.2063delA c.3582del 10bp c.2688delT c.3560delG g.3599del34bp g.3715ins2bp c.3141delC
Predicted protein
Exon
Diagnosis
References
–
5
DGI-III
32
p.S624TfsX687 p.S640TfsX671 p.S640TfsX671 p.S758AfsX554 p.S842TfsX471 – – – – – – –
5 5 5 5 5 5 5 5 5 5 5 5
DD-II DD-II DD-II DGI-II DGI-II DD-II DGI-III DGI-II DGI-II DGI-III DGI-III DD-II
26 26 17 26 26 17 17 50 50 44 44 51
journal of dentistry 40 (2012) 542–548
mutations led to the phenotypes of dentinogenesis imperfecta type II or dentine dysplasia, whereas in frame mutations did not cause observable clinical changes.17
4.
Conclusions
Even though the sophisticated methods used currently for molecular analysis of the DSPP gene precisely define types and localizations of DSPP mutations, the direct correlation between the specific mutation and manifested clinical symptoms is not possible yet. Possibly, there are many other contributing factors together with environmental ones52 which, acting in concert with genetics, influence the final phenotype observed in the patients diagnosed with dentinogenesis imperfecta type II or dentine dysplasia. Clinical diagnosis of dentinogenesis imperfecta type II and dentine dysplasia includes recognition of a cluster of symptoms that accompany this hereditary dentine disorder(s), although individual cases differ one from another in regard to teeth discolouration, cervical region constriction, presence of pulp stones, severity of teeth attrition or coexistence of periapical inflammation. Thus detailed progressive clinical evaluations of probants and their multi-generation families seem critical. The histological study of mutated teeth, both deciduous and permanent, could bring more data as well. It is not clear yet whether the mutated DSPP molecule is processed in ER and subsequent organelles prior to final release into the extracellular matrix of dentine or if a change in the chemical properties of DSPP results in aggregation of unprocessed DSPP in the cytosol of odontoblasts. It might be speculated that the most frequent mutations, which appear in the code for the signal peptide and/or three first amino acids of the coding sequence of DSPP, impact on the transport and processing of the mutated protein, leading to it becoming trapped and blocking cellular events in the rough ER. This might eventually affect translation of DSPP and also other proteins resulting in disrupted dentine formation. However, the reading frameshift mutations of the DPP code change the properties of DPP, which may disrupt the entire process of dentine maturation, eventually resulting in the severe phenotype of dentinogenesis imperfecta type II and/or dentine dysplasia. There is thus considerable scope for further research at the cellular level to determine the mechanistic effects of DSPP mutations. So far researchers mostly speculate about the potential protein formation, predicting the final protein sequence from the analysis of the sequence of mutated DNA, although the most specific data would come from the direct co-evaluation of both the mutated gene and protein. Such a detailed analysis should precisely answer the query about what ‘‘protein’’ is formed from the specifically mutated gene. The use of DSPP gene sequencing, especially for the repetitive sequence of the DPP code, is important in the clinical diagnosis of patients with dentinogenesis imperfecta type II/III and dentine dysplasia, but our understanding of the cellular fate of mutated DSPP is still unclear. Such knowledge may help to underpin future molecular therapies for these patients and thus comprehensive research on the cellular fate and processing of both normal and mutated DSPP is required.
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Acknowledgement The authors acknowledge the helpful advice of Dr. AJ Smith and Mr. Michael Liley.
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