De novo mutation in the DSPP gene associated with dentinogenesis imperfecta type II in a Japanese family

Dentinogenesis imperfecta (DGI) type II is one of the most common dominantly inherited dentin defects, in which both the primary and permanent teeth are affected. Here, we report a Japanese family with autosomal‐dominant DGI type II, including both molecular genetic defects and pathogenesis with histological analysis. Mutation analysis revealed a mutation (c.53T>A, p.V18D, g.1192T>A) involving the second nucleotide of the first codon within exon 3 of the dentin sialophosphoprotein (DSPP) gene. This mutation has previously been reported in a Korean family. Thus far, 24 allelic DSPP mutations have been reported, and this is the seventh mutation involving the DSPP V18 residue. Among those, only one other was shown to be caused by a de novo mutation, and that mutation also affected the V18 amino acid residue. The DSPP V18 residue is highly conserved among other mammalian species. These findings thus suggest that the V18 amino acid might be a sensitive mutational hot spot, playing a critical role in the pathogenesis of DGI.     

Refined mapping of the human dentin sialophosphoprotein (DSPP) gene within the critical dentinogenesis imperfecta type II and dentin dysplasia type II loci

Dentinogenesis imperfecta type II and dentin dysplasia type II are diseases resulting in abnormal dentin formation, which have been mapped to overlapping regions of human chromosome 4q defined by markers D4S2691 and D4S2692 (6.6 cM) and D4S3291 and SPP1 (14.1 cM), respectively. Recently, two of the major non-collagenous proteins of dentin, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP, phosphophoryn) have been shown to be encoded by a single gene, termed dentin sialophosphoprotein (DSPP), which has been mapped to human chromosome 4. The purpose of this study was to perform refined mapping of DSPP related to these disease loci by gene content mapping, as well as to place the DSPP gene on the physical map of human chromosome 4 by sequence tagged site (STS) content mapping. Human genomic DSPP clones were isolated, and gene content mapping performed with specific primers for dentin matrix protein 1 (DMP1), bone sialoprotein (BSP) and osteopontin (secreted phosphoprotein 1, SPP1). STS content mapping was then performed with flanking STS markers to these dentin/bone gene loci. Our results demonstrate that the DSPP and DMP1 genes are within a maximum distance of 110 kb. Both DSPP and DMP-1 have been placed on the physical map of human chromosome 4 within the interval defined by markers D4S564 and D4S1292. DSPP is thereby strengthened as a candidate gene for both DGI-II and DD-II.     

牙本质发育不全Ⅱ型的遗传异质性研究

目的：明确牙本质涎磷蛋白(dentin sialophosphoprotein，DSPP)是否为该家系的致病基因，并对该家系做进一步的基因定位研究。方法：通过DNA测序方法对DSPP基因进行突变检测，用位于4q21区域的7个微卫星位点对家系进行遗传连锁分析。结果：测序结果显示DSPP在该家系中不存在突变，基因定位研究表明致病基因在该家系位于IMS1534和DSPP之间。结论：DSPP在该家系不是致病基因，牙本质发育不全Ⅱ型存在遗传异质性。     

遗传性牙本质发育不全Ⅱ型DSPP基因新突变

目的分析我国遗传性牙本质发育不全Ⅱ型(dentinogenesisimperfectatypeⅡ,DGI-Ⅱ)患者DSPP基因突变特征,从分子水平探讨DGI-Ⅱ的发病机制。方法抽提2个汉族遗传性牙本质发育不全Ⅱ型家系患者外周静脉血基因组DNA,应用聚合酶链反应及DNA测序技术,结合序列分析方法,对2个家系共13名家庭成员的DSPP基因1～4号外显子及其邻近序列进行突变分析。结果家系A中的患者在DSPP的第4外显子发生Asn164Tyr突变;家系B的患者在DSPP第4外显子发生Cys159Trp突变。结论这两个突变系是国内外尚未报道的新突变。     

Hyperfibers and vesicles in dentin matrix in dentinogenesis imperfecta (Dl) associated with osteogenesis imperfecta (01)

Dentin matrix of deciduous teeth from two patients affected by dentinogenesis imperfecta (DI) associated with types IB and IVB osteogenesis imperfecta (Ol) displayed previously undescribed structures in transmission electron microscopic examination. Vesicles were seen in dentin of both patients, and abnormally thick collagen fibers (hyperfibers) were found in dentin of the patient with the rare type IB OI. Both vesicles and hyperfibers were situated in abnormal, atubular areas of dentin. Matrix vesicles, which have normally been identified in mantle dentin only, were abundant in selected areas of the affected dentin, thereby supporting the concept that dentin matrix in OI is elaborated by successive cell generations. The hyperfibers, not previously described in either normal or abnormal human dentin, have possibly been formed by fusion of several collagen fibers. Further ullrastructural studies of dentin in DI with OI may help to clarify the marked clinical variation in teeth of patients affected by OI.     

Mild forms of dentinogenesis imperfecta in association with osteogenesis imperfecta as characterized by light and transmission electron microscopy

Osteogenesis imperfecta (OI) results from various gene mutations leading to defects in type I collagen, which is the major component of both bone and dentin. Yet dentinogenesis imperfecta (DI) is found only in half of the patients with OI. Here we document patients from three families with OI and DI lacking the clinical and radio-graphic features of DI in permanent teeth. However, light and transmission electron microscopic studies of dentin of deciduous and permanent teeth revealed various changes in the morphology of the dentinal tubules and collagen fibers. In one family, diagnosis of DI preceded that of OI. The grade of severity of dentinal manifestations in patients with OI apparently forms a continuum from normal dentin structure to severe DI. and the marked difficulty in diagnosing mild DI may have led to underestimating its frequency. Furthermore, patients with DI should be carefully examined for the possible presence of OI.     

Familial tarda type osteogenesis imperfecta with dentinogenesis imperfecta Type I. Case report

This paper presents a case of dentinogenesis imperfecta Type I occurring in a patient with familial tarda type osteogenesis imperfecta. The investigation and management of this patient is described.     

Role of the NH2‐terminal fragment of dentin sialophosphoprotein in dentinogenesis

Dentin sialophosphoprotein (DSPP) is a large precursor protein that is proteolytically processed into a NH₂‐terminal fragment [composed of dentin sialoprotein (DSP) and a proteoglycan form (DSP‐PG)] and a COOH‐terminal fragment [dentin phosphoprotein (DPP)]. In vitro studies indicate that DPP is a strong initiator and regulator of hydroxyapatite crystal formation and growth, but the role(s) of the NH₂‐terminal fragment of DSPP (i.e. DSP and DSP‐PG) in dentinogenesis remain unclear. This study focuses on the function of the NH₂‐terminal fragment of DSPP in dentinogenesis. Here, transgenic (Tg) mouse lines expressing the NH₂‐terminal fragment of DSPP driven by a 3.6‐kb type I collagen promoter (Col 1a1) were generated and cross‐bred with Dspp null mice to obtain mice that express the transgene but lack the endogenous Dspp (Dspp KO/DSP Tg). We found that dentin from the Dspp KO/DSP Tg mice was much thinner, more poorly mineralized, and remarkably disorganized compared with dentin from the Dspp KO mice. The fact that Dspp KO/DSP Tg mice exhibited more severe dentin defects than did the Dspp null mice indicates that the NH₂‐terminal fragment of DSPP may inhibit dentin mineralization or may serve as an antagonist against the accelerating action of DPP and serve to prevent predentin from being mineralized too rapidly during dentinogenesis.     

Bilateral cracked permanent first molars in dentinogenesis imperfecta type II: A case report

Dentinogenesis imperfecta type II (DI-II) is an inherited mesodermal condition affecting the primary and permanent dentition. There is often cracking and loss of enamel and the subsequent rapid attrition of exposed dentin. This report presents a 14-year-old boy with DI-II, specifically with an unusual case of cracked bilateral maxillary and mandibular first molars. Fractures involved enamel, dentin, and cementum.     

Functional splicing assay of DSPP mutations in hereditary dentin defects

Oral Diseases (2011) 17 , 690–695 Objective: Dentin sialophosphoprotein (DSPP) gene mutations have been identified in isolated hereditary dentin defects; however, the genotype–phenotype correlations are poorly understood. We performed in vitro splicing assays to test the hypothesis that DSPP mutations in splice junctions as well as proposed missense/nonsense mutations experimentally result in aberrant pre‐mRNA splicing. Materials and methods: The genomic fragment of the human DSPP gene was cloned into the pSPL3 splicing vector, and previously reported as well as informative de novo mutations were then introduced by PCR mutagenesis. The COS‐7 cells were transfected with each plasmid vector, and total RNA was isolated. RT‐PCR result was analyzed, and the band intensity of the product was calibrated using ImageJ. Results: The predictions by others of exon 3 skipping in specific DSPP mutations have been validated and a cryptic splicing donor site has been identified. However, the degree of mutational effect on pre‐mRNA splicing varied considerably depending on the changed nucleotide. Conclusions: The predictions of exon 3 skipping in specific DSPP mutations have been validated, and a cryptic splicing donor site has been identified. Our data may provide insight into the contribution of DSPP mutations in the pathogenesis and genotype–phenotype correlations of hereditary dentin defects.     

遗传性乳光牙本质家系致病基因的染色体定位

目的　研究一个在天津塘沽地区发现的遗传性乳光牙本质回族家系致病基因是否与染色体 4q2 1连锁。方法　提取该家系 1 3名成员的外周血DNA ,选择染色体 4q2 1上的 8个短串联重复序列多态性标记 (shorttandemrepeatpolymorphisms ,STRPs)做荧光标记PCR等位片段分析 ,用lod连锁分析法分析该家系致病基因位点与上述 8个STRPs的连锁关系。结果　得到 1 3名个体 8个位点的基因型和单体型 ,连锁分析结果显示 :8个STRPs的最大lod值均大于 0 ,其中 5个STRPs的lod值大于 1。结论　该家系致病基因定位在染色体 4q2 1上 ,表明中国回族人和报道的欧美人的DGI Ⅱ的基因座位是一致的     

The non-collagenous dentin matrix proteins are involved in dentinogenesis imperfecta type II (DGI-II)

Dentinogenesis Imperfecta type II (DGI-II) is a localized form of mesodermal dysplasia of the dentin affecting both the primary and permanent dentitions. This is an autosomal-dominant disease in which there is a disorder in dentin mineralization. Several studies have localized DGI-II to human chromosome 4 in the region 4q 12-21. Many ECM genes-such as OPN, DMP1, DMP2, DMP3 (DSPP), and BSP-have been mapped to the same locus. Biochemical studies indicated that dentin phosphophoryn (DMP2) might be a candidate gene in DGI-II. In this study, we have used histological and RFLP analyses of tissues from a DGI-II-affected patient, as compared with two normal controls, to determine if DMP1, 2, or 3 was linked to DGI-II. The histology of the affected tooth was very different in the DGI-II patient as compared with the normals. In particular, the dentinal tubules in the DGI-II patient were very irregular, which could be the result of perturbations in the process of dentin formation. Patient and control DNA samples were digested with EcoRI or PstI and Southern-hybridized with the DMP1, DMP2, and DMP3 cDNAs. Few differences in the restriction pattern were observed between affected and normal samples for DMP1 and DMP3-3' region (phosphophoryn-like sequences) probes. On the other hand, DMP2 showed a dramatic shift in the restriction pattern in DGI-II. This study suggests that the different restriction enzyme digestion profiles of the DNA from the DGI-II patient, as probed by DMP2, might be related to the defective mineralization of dentin in DGI-II.     

小鼠牙胚、软骨组织中牙本质涎磷蛋白基因的克隆

目的：从小鼠牙胚、软骨组织中克隆牙本质涎磷蛋白基因（dentin sialophosphoprotein,DSPP)。方法：采用RT-PCR方法,从小鼠牙胚及软骨组织中克隆DSPP基因片段,将所得片段装入载体pGEM-TEasyVector进行序列测定。结果：从两种组织中均获得719bp的特异性片段,序列分析表明,与已发表的DSPP序列99.7%同源。结论：成功地从小鼠牙胚。软骨中克隆到DSPP基因部分序列。结果提示：除牙齿组织外,DSPP还可在软骨中表达,它可能并非是检测成牙本质细胞的特异性指标。     

Histomorphologic investigations of dentinogenesis in incisors of offspring of cyclophosphamide-treated pregnant rats

abstract — Eight days old offspring of six rats treated with single doses of 40 mg/kg and 50 mg/kg of cyclophosphamide on the 20th day of pregnancy constituted two experimental groups. Group 1 (40 mg/kg) comprised 21 animals, Group 2 (50 mg/kg) 14 animals. Histomorphologic investigations of the maxillary and mandibular incisors revealed the following changes: (1) dental constrictions, (2) niche-like dentinal defects, (3) scalloping of the amelodentinal junction, (4) incremental line in the pre-experimental tooth segment, (5) external resorptions, and (6) diffuse edema in the pre-experimental part of pulp. Constrictions and niches qualitatively concurred with those previously observed in adult rats of the same strain, but the lesion frequency was essentially higher in the offspring. Further, the additional change of external resorptions was evinced by the offspring incisors, and changes only rarely observed in adults, such as scalloping of the dentinoenamel junction and a dentinal incremental line, constituted frequent findings in the offspring material.     

Identification of the DSPP mutation in a new kindred and phenotype-genotype correlation

Oral Diseases (2011) 17 , 314–319 Objective: Hereditary dentin defects can be grouped into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia. Tooth enamel is considered normal in patients with hereditary dentin defects, but is easily worn down and fractured due to DSPP mutation‐induced altered dentin properties. The purposes of this study were to identify genetic cause of a family with type II DGI and enamel defects. Materials and methods: We identified a family with type II DGI and a unique form of hypoplastic enamel defect affecting occlusal third of the crown. Family members were recruited for the genetic analysis and DNA was obtained from peripheral whole blood. Results: Mutational analysis revealed a T to A transversion in exon 3 of the DSPP (c.53T>A, p.V18D). Haplotype analysis showed that the same mutation arose separately in two different families having DGI with similar enamel defects, indicating that this phenotype is associated with this specific DSPP mutation. Clinical features suggest that enamel formation was affected in the affected individuals during early amelogenesis, in addition to the dentin defect. Conclusions: We observed that a DSPP gene mutation not only influences dentinogenesis but also affects early stage amelogenesis.     

Glycosaminoglycans of EDTA soluble and insoluble dentin in dentinogenesis imperfecta type I.

A study of glycoasminoglycans (GAGs) in dentinogenesis imperfecta Type I (DI I) revealed increased GAG in DI I EDTA soluble dentin as compared to controls. EDTA insoluble GAG contained less GAG than age-matched controls. The role of GAG in dentin pathosis is discussed.