Novel dentin phosphoprotein frameshift mutations in dentinogenesis ...

© 2010 John Wiley & Sons A/S

Clin Genet 2011: 79: 378–384 Printed in Singapore. All rights reserved

CLINICAL GENETICS doi: 10.1111/j.1399-0004.2010.01483.x

Short Report

Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II Lee K-E, Kang H-Y, Lee S-K, Yoo S-H, Lee J-C, Hwang Y-H, Nam KH, Kim J-S, Park J-C, Kim J-W. Novel dentin phosphoprotein frameshift mutations in dentinogenesis imperfecta type II. Clin Genet 2011: 79: 378–384. © John Wiley & Sons A/S, 2010 The dentin sialophosphoprotein (DSPP) gene encodes the most abundant non-collagenous protein in tooth dentin and DSPP protein is cleaved into several segments including the highly phosphorylated dentin phosphoprotein (DPP). Mutations in the DSPP gene have been solely related to non-syndromic form of hereditary dentin defects. We recruited three Korean families with dentinogenesis imperfecta (DGI) type II and sequenced the exons and exon–intron boundaries of the DSPP gene based on the candidate gene approach. Direct sequencing of PCR products and allele-specific cloning of the highly repetitive exon 5 revealed novel single base pair (bp) deletional mutations (c.2688delT and c.3560delG) introducing hydrophobic amino acids in the hydrophilic repeat domain of the DPP coding region. All affected members of the three families showed exceptionally rapid pulp chambers obliteration, even before tooth eruption. Individuals with the c.3560delG mutation showed only mild, yellowish tooth discoloration, in contrast to the affected individuals from two families with c.2688delT mutation. We believe that these results will help us to understand the molecular pathogenesis of DGI type II as well as the normal process of dentin biomineralization.

K-E Leea , H-Y Kanga , S-K Leea , S-H Yoob , J-C Leec , Y-H Hwanga , KH Nama , J-S Kimb , J-C Parka and J-W Kima,d a Department of Cell and Developmental Biology & Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea, b Department of Pediatric Dentistry, School of Dentistry, Dankook University, Chung Nam, Korea, c Seoul Children’s Dental Center, Seoul, Korea, and d Department of Pediatric Dentistry & Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea

Key words: dentin dysplasia – dentin sialophosphoprotein – dentinogenesis imperfecta – frameshift mutation Corresponding author: Dr Jung-Wook Kim, Department of Molecular Genetics, and Department of Pediatric Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, 275-1 Yongon-dong, Chongno-gu, Seoul 110-768, Korea. Tel.: +82 2 2072 2639; fax: +82 2 744 3599; e-mail: [email protected] Received 18 April 2010, revised and accepted for publication 3 June 2010

Dentin is one of the main components of the tooth and has a unique structure. The mechanical characteristics and organic content of dentin are similar to those of the bone, but long odontoblast processes remain in the dentin matrix and function as a nociceptive receptor and a nutritional supplier (1). Tooth enamel, the hardest tissue in the human body, needs to be well supported by dentin in order to function properly without easy fracture or attrition. Therefore, defective dentin not only has its own poor mechanical properties, but it also exerts a detrimental effect on the enamel. 378

Hereditary dentin defect has been categorized by dentinogenesis imperfecta (DGI types I, II, and III) and dentin dysplasia (DD types I and II) (2–3). DGI type I (MIM 166240) is a syndromic dental phenotype of osteogenesis imperfecta that shares similar features with DGI type II (MIM #125490), but the penetrance and expressivity are the variables. DGI type II is characterized by brown opalescent dentition, a bulbous crown shape, and pulpal obliteration. DGI type III (MIM #125500) is now believed to be a more severe form of DGI type II, sometimes showing multiple pulp exposures and shell-like teeth. In contrast, DD

Novel dentin phosphoprotein frameshift mutations

type II (MIM #125420) is similar to DGI type II during deciduous dentition formation, but the permanent dentition has only minimal discoloration and a thistle-tube shaped pulp chamber with pulp stones. Linkage studies (4–6) and mutational analyses (7–9) have shown that these three disease entities are allelic with varying degrees of severity. Dentin sialophosphoprotein (DSPP) is the most abundant non-collagenous protein in dentin (10). DSPP is synthesized as a single protein by odontoblasts and BMP1 cleaves the DSPP protein into two major fragments: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) (11, 12). Genetic studies have revealed mutations in the DSPP gene in the DGI types II and III as well as in DD type II (13–17). Even though other candidate genes for non-syndromic hereditary dentin defects have been proposed (18), such as matrix extracellular phosphoglycoprotein (MEPE ) and integrin-binding sialoprotein (IBSP ), only DSPP gene mutations have been identified to date. With the progress in the sequencing of the highly repetitive DPP region, net-1 frameshift mutations resulting in hydrophobic C-termini have been reported by two groups (19–21); however, the mechanism of molecular pathogenesis for hereditary dentin defects remains unknown. In this study, we report novel mutations (c.2688delT and c.3560delG) in the DPP region and their clinical phenotype in three Korean families. Materials and methods Enrollment of human subjects

Three families with dentinogenesis imperfecta were recruited for this study. The study protocol was independently reviewed and approved by the institutional review board at the Seoul National University Dental Hospital. The experiments were performed with the understanding and written consent of each participating subject according to the Declaration of Helsinki. Primer design, polymerase chain reaction, and DNA sequencing

Genomic DNA was isolated from peripheral blood using the QuickGene DNA whole blood kit S with QuickGene-Mini80 equipment (Fujifilm, Tokyo, Japan). Polymerase chain reaction (PCR) amplifications and DNA sequencing were performed according to the previously described conditions for the non-repeat (7) and repeat domains (20). PCR reactions were performed using HiPi DNA polymerase premix (ElpisBio, Daejeon, Korea),

and the PCR products were purified according to the supplied protocol with the PCR Purification Kit (ElpisBio). DNA sequencing was performed at the DNA sequencing center (Macrogen, Seoul, Korea). All nucleotide numbering was counted from the A of the ATG translational initiation codon of the human DSPP reference sequence (NM_014208.3). Allele-specific cloning of DSPP and sequencing

In order to sequence individual alleles of the DPP region, amplified PCR products of exon 5 of the DSPP gene were cloned as previously described (20) using the Topcloner PCR cloning kit (Enzynomics, Seoul, Korea). Individual clones were sequenced and analyzed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Results Mutation results

Mutational analysis revealed that all probands had a heterozygous single nucleotide deletion in exon 5 of the DSPP gene. Probands 1 (from family 1) and 2 (from family 2) had the same mutation (c.2688delT) (Fig. 1c–e) and haplotype analysis revealed that this mutation originated from a common ancestor (identical by descent) by sequencing nearby SNPs (IVS3-33, IVS3-29, c.727, c.897; data not shown). Proband 3 (from family 3) had a G deletion (c.3560delG) near the carboxy-terminus of the DSPP protein (Fig. 3b–d). These mutations cause a frameshift in the coding sequence, which replaces the acidic SSD repeats in the DPP region with novel, highly hydrophobic amino acids. The wildtype DSPP gene codes 1301 amino acids. The c.2688delT mutant allele would produce 1265 amino acid DSPPs without deletion, but the deletion introduced 372 hydrophobic amino acids instead of 361 original amino acids, and the c.3560delG mutant allele would produce 1259 amino acid DSPPs without deletion, but the deletion introduced 126 hydrophobic amino acids instead of 115 original amino acids. Our mutation nomenclature is based on the current DSPP reference sequence (NM_14208.3), but the exact sequence of each frameshift is deposited in GenBank (GU983874 and GU983875). Clinical findings

Proband 1 (III:5; Fig. 1a) was 6.5 years old when he presented to the Pediatric Dental Clinic at the Seoul National University Dental Hospital. His dentition was brown to bluish opalescent, and his 379

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Fig. 1. Pedigrees and mutational analysis. (a) Pedigree of family 1. (b) Pedigree of family 2. (c) DNA sequencing chromatogram of the PCR amplification product from the proband of family 1. (d) DNA sequencing chromatogram of the cloned normal allele. (e) DNA sequencing chromatogram of the cloned mutated allele (c.2688delT).

molars had a characteristic bulbous crown form (Fig. 2b). The pulp chambers were obliterated almost completely even before tooth eruption (Fig. 2a). Proband 2 (III:1, Fig. 1b) was a 9-yearold boy who exhibited dark brown opalescent permanent dentition (Fig. 2c). The other clinical and radiological phenotypes of proband 2 were similar to those of proband 1 (Fig. 2d). Proband 3 (IV:2; Fig. 3a) was a 10-year-old boy who had bulbous molar crowns. His pulp chambers were obliterated almost completely even before tooth eruption, as observed in the other two probands (Fig. 4d). However, the permanent 380

dentition had only a mild yellowish discoloration (Fig. 4a–c). All members of the three families denied other symptoms such as bone fragility or hearing loss. Discussion

The DPP coding region has a highly repetitive sequence, which is proposed to originate from the primodial 9 bp sequence (AGC AGC GAC) (22). DPP is a highly hydrophilic protein that contains aspartic and phosphoserine acids in more than 85% of its amino acid composition. DPP interacts with collagen fibrils in the dentin matrix and nucleates


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Fig. 2. Clinical photos and panoramic radiographs. (a) A dental panoramic radiograph of the proband of family 1, taken at the age of 6.5 years. (b) A frontal photograph of the proband of family 1, taken at the age of 6.5 years. (c) A frontal photograph of the proband of family 2, taken at the age of 9 years. (d) Dental panoramic radiograph of the proband of the family 2, taken at the age of 9 years.

mineral crystallites through binding calcium ions via its highly acidic nature (23). In this study, we identified two novel frameshift mutations in the DPP coding region in three Korean families with DGI type II. A net-1 bp deletional mutation causing a frameshift in the DPP repeat domain does not result in an early translational stop codon due to its unique repetitive nucleotide sequence, but instead introduces novel highly hydrophobic amino acids (mainly alanine, valine, threonine, isoleucine) in the place of hydrophilic SSD repeat domain.

The exact function of DSPP-derived proteins in biomineralization is not well known. However, a recent study of a DSP-expressing transgenic mouse on a DSPP-null genetic background determined that DSP and DPP have distinct roles in dentin mineralization: DSP regulates the initiation of dentin mineralization, and DPP is involved in the maturation of dentin mineralization (24). The frameshift mutations in the DPP repeat domain would result in DSPP protein misfolding or trapping into the rER membrane due to the hydrophobic amino acid repeat, which might induce endoplasmic reticulum stress. This could 381

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Fig. 3. Pedigree and mutational analysis. (a) Pedigree of family 3. (b) A DNA sequencing chromatogram of the PCR amplification product from the proband. Reverse primer was used for the sequencing reaction and the chromatogram is a result of a computerderived inversion. (c) A DNA sequencing chromatogram of the cloned normal allele. (d) A DNA sequencing chromatogram of the cloned mutated allele (c.3560delG).

influence the cell’s capability to produce and process protein, reducing the amount of mutant DSPP as well as the amount of normal DSPP and/or other critical proteins that are involved in dentin mineralization (20). This reduction in the level of dentin matrix proteins would be less than the amount that would result from simple haploinsufficiency. Another possibility is that the mutant protein is secreted in the dentin matrix. The mutant protein may have a reduced capability to interact with 382

collagen and to bind to calcium ions. This may result in a defect in the dentin mineralization. One of the identified mutations in this study is the most 3 mutation in the DPP domain, a mutation that introduces only 126 novel hydrophobic amino acids, thus the mutational effect would be milder than those of the other frameshift mutations. The mutant protein would retain partial capability to interact with collagen and to bind to calcium ions. This could explain the less severe clinical phenotype (only a mild, yellowish discoloration).


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Fig. 4. Clinical photos and panoramic radiograph. (a) A frontal photograph of the proband of family 3, taken at the age of 10 years. (b) A maxillary occlusal photograph of the proband of the family 3, taken at the age of 10 years. (c) A mandibular occlusal photograph of the proband of family 3, taken at the age of 10 years. (d) A dental panoramic radiograph of the proband of family 3, taken at the age of 10 years.

However, the length of the mutant hydrophobic amino acid chain does not correlate with the severity of the disease in all cases. The frameshift mutations occurred in the anterior region of DPP showed a DD type II clinical phenotype, a less severe form of hereditary dentin defects, in spite of a longer hydrophobic amino acid chain than the other frameshift mutations associated with DGI type II (19, 20). Furthermore, a single bp

deletional mutation (c.3141delC) was identified in a family that had an overlapping phenotype between DD type II and DGI type II (21). Because the location of the mutation was in the region that was previously associated with only DGI type II, the existence of a genetic modifier closely linked to the DSPP gene may explain this phenomenon (21). Even though the clinical phenotypes were varied in the three families, the pulp chambers in 383

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these families were obliterated quickly and almost completely, even before tooth eruption. Mutations in the DSP region resulting in DGI type II generally also lead to the obliteration of the pulp chamber, but the obliteration is not as rapid and the almost complete obliteration before tooth eruption has not usually occurred. The DPP protein would be intact when the mutation occurred in the DSP region, but the mutant DPP protein would be secreted when the mutation occurred in the DPP region. Mutant DPP protein may lose its functional role in the maturation of dentin mineralization. This would reduce the time necessary for maturation, thus less-mineralized dentin would be made at a high speed. In conclusion, we have identified novel single bp deletional DSPP mutations in three Korean families with DGI type II. Molecular genetic etiology and its related clinical features will add evidence to help us to understand the genotype–phenotype correlations of hereditary dentin defects. Further mutational analyses and functional characterization of DSPP and its cleaved segments need to be conducted in order to unravel the processes involved in normal and pathological dentin biomineralizations.

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Acknowledgements We would like to thank all the family members for their cooperation. This work was supported by a grant from the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (M10646010003-08N4601-00310), the Science Research Center grant to Bone Metabolism Research Center (2009-0063266) funded by the Korean Ministry of Education, Science and Technology, and a grant of the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084701).

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Competing interest

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