Mutational hot spot in the DSPP gene causing dentinogenesis imperfecta type II

The current system for the classification of hereditary defects of tooth dentin is based upon clinical and radiographic findings and consists of two types of dentin dysplasia (DD) and three types of dentinogenesis imperfecta (DGI). However, whether DGI type III should be considered a distinct phenotype or a variation of DGI type II is debatable. In the 30 years since the classification system was first proposed, significant advances have been made regarding the genetic etiologies of inherited dentin defects. DGI type II is recognized as an autosomal dominant disorder with almost complete penetrance and a low frequency of de novo mutations. We have identified a mutation (c.52GT, p.V18F) at the first nucleotide of exon 3 of the DSPP (dentin sialophosphoprotein) gene in a Korean family (de novo) and a Caucasian family. This mutation has previously been reported as causing DGI type II in a Chinese family. These findings suggest that this mutation site represents a mutational hot spot in the DSPP gene. The clinical and radiographic features of these two families include the classic phenotypes associated with both DGI type II and type III. Finding that a single mutation causes both phenotypic patterns strongly supports the conclusion that DGI type II and DGI type III are not separate diseases but rather the phenotypic variation of a single disease. We propose a modification of the current classification system such that the designation hereditary opalescent dentin or DGI type II should be used to describe both the DGI type II and type III phenotypes.     

A novel splicing mutation alters DSPP transcription and leads to dentinogenesis imperfecta type II

Dentinogenesis imperfecta (DGI) type II is an autosomal dominant disease characterized by a serious disorders in teeth. Mutations of dentin sialophosphoprotein (DSPP) gene were revealed to be the causation of DGI type II (DGI-II). In this study, we identified a novel mutation (NG_011595.1:g.8662T>C, c.135+2T>C) lying in the splice donor site of intron 3 of DSPP gene in a Chinese Han DGI-II pedigree. It was found in all affected subjects but not in unaffected ones or other unrelated healthy controls. The function of the mutant DSPP gene, which was predicted online and subsequently confirmed by in vitro splicing analysis, was the loss of splicing of intron 3, leading to the extended length of DSPP mRNA. For the first time, the functional non-splicing of intron was revealed in a novel DSPP mutation and was considered as the causation of DGI-II. It was also indicated that splicing was of key importance to the function of DSPP and this splice donor site might be a sensitive mutation hot spot. Our findings combined with other reports would facilitate the genetic diagnosis of DGI-II, shed light on its gene therapy and help to finally conquer human diseases.     

Paraneoplastic limbic encephalitis presenting as a neurological emergency: a case report

Introduction

Dentin dysplasia is a rare hereditary disturbance of dentin formation characterized by defective dentin development with clinically normal appearing crowns, severe hypermobility of teeth and spontaneous dental abscesses or cysts. Radiographic analysis shows obliteration of all pulp chambers, short, blunted and malformed or absent roots and peri-apical radiolucencies of non carious teeth.

Case presentation

We present a case of dentin dysplasia type I in a 12-year-old Iranian boy, and the clinical, radiographic and histopathologic findings of this condition and treatment are described.

Conclusions

There are still many inconclusive issues in the diagnosis and management of patients with dentin dysplasia. The diagnostic features of this rare disturbance will remain incompletely defined until additional cases have been described. Early diagnosis of the condition and initiation of effective regular dental treatments may help these patients to prevent or delay loss of dentition.     

N-terminal Dentin Sialoprotein fragment induces type I collagen production and upregulates dentinogenesis marker expression in osteoblasts

Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein (DSPP) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly⁴⁶²-Asp⁴⁶³, resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen, Runx2, Osterix and ATF4. Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes.     

New genetic variants identified in donkey's milk whey proteins

Novel genetic variants for donkey milk lysozyme and -lactoglobulins I and II have been identified by the combined use of peptide mass mapping and sequencing by tandem mass spectrometry in association with database searching. The novel donkey lysozyme variant designated as lysozyme B (Mr 14,631 Da) differed in three amino acid exchanges, N⁴⁹ D, Y⁵² S, and S⁶¹ N, from the previously published sequence. Three novel genetic variants for donkey -lactoglobulins were identified. One of them is a type -lactoglobulin I with three amino acid exchanges at E³⁶ S, S⁹⁷ T, and V¹⁵⁰ I (-lactoglobulin I B, Mr 18,510 Da). The two others are type -lactoglobulins II with two amino acid exchanges at C¹¹⁰ P and M¹¹⁸ T (-lactoglobulin II B, Mr 18,227 Da) and with three amino acid exchanges at D⁹⁶ E, C¹¹⁰ P, and M¹¹⁸ T (-lactoglobulin II C, Mr 18,241 Da). All these primary structures are closely related to those of homologous proteins in horse milk (percent identity >96%).     

β-Thalassemia mutations in Singapore — a strategy for prenatal diagnosis

The strategy for early prenatal diagnosis of -thalassemia in Singapore by direct detection of the mutant -globin gene requires the spectrum of mutations producing the disorder in this population to be characterized. We analyzed 134 -thalassemia alleles from Singapore by specific oligonucleotide hybridization after DNA amplification, using a nonradioactive enhanced chemiluminescence detection system. The mutations were identified in 90% of the alleles using five oligonucleotide probes for the following mutations: codons 41/42 (deletion -TCTT), IVS II nt 654 (CT), codon 17 (AT), IVS I nt 5 (GC), and -28 TATA box (AG). Together with the strategy of direct sequencing, a total of 97% of the mutations were identified. In the Chinese subpopulation, 97% of the mutations were detected by the oligonucleotide probes. Using just four oligonucleotide probes would identify 96% of the mutations, and 76% of the mutations were accounted for by codon 41/42 (-TCTT) and IVS II nt 654 (CT) mutations. Thus in this subpopulation early prenatal diagnosis would be possible in virtually all the affected families.     

Spondyloepiphyseal dysplasia and precocious osteoarthritis in a family with an Arg75→Cys mutation in the procollagen type II gene (COL2A1)

Direct sequencing of polymerase chain reaction (PCR)-amplified genomic DNA from a patient with spondyloepiphyseal dysplasia and precocious osteoarthritis revealed a single-base change in exon 11 of the type II procollagen gene (COL2A1), which produces an Arg Cys mutation in one allele. The proband is a member of a large Chilean kindred presenting with chondrodysplasia of the hips, knees, shoulders, elbows, and spine associated with severe, early-onset osteoarthritis. All affected individuals exhibit mildly short stature; in addition, five out of seven affected family members display shortened metacarpals or metatarsals. DNA from affected and unaffected family members was PCR-amplified and analysis of restriction digests of the products determined that the mutation segregated with the disease with a lod score of 2.2 at zero recombination. The mutation, which resides in the triple-helical region of type II procollagen at amino acid position 75, is the second example of an ArgCys mutation in the COL2A1 gene in heritable cartilaginous disease and is the first example of a point mutation in the amino terminal region of the 1(II) chain, that results in a spondyloepiphyseal dysplastic phenotype.     

Functional size of Photosystem II determined by radiation inactivation

The functional size of Photosystem II (PS II) was investigated by radiation inactivation. The technique provides an estimate of the functional mass required for a specific reaction and depends on irradiating samples with high energy -rays and assaying the remaining activity. The analysis is based on target theory that has been modified to take into account the temperature dependence of radiation inactivation of proteins. Using PS II enriched membranes isolated from spinach we determined the functional size of primary charge separation coupled to water oxidation and quinone reduction at the Q_B site: H₂O (Mn)₄ Y_z P680 Pheophytin Q phenyl-p-benzoquinone. Radiation inactivation analysis indicates a functional mass of 88 ± 12 kDa for electron transfer from water to phenyl-p-benzoquinone. It is likely that the reaction center heterodimer polypeptides, D1 and D2, contribute approximately 70 kDa to the functional mass, in which case polypeptides adding up to approximately 20 kDa remain to be identified. Likely candidates are the and subunits of cytochrome b ₅₅₉and the 4.5 kDa psbI gene product.Abbreviations Cyt cytochrome - PS Photosystem - P680 primary electron donor of Photosystem II - Q_A primary quinone acceptor of Photosystem II - Q_B secondary quinone acceptor of Photosystem II - Y_z tyrosine donor to P680     

Three novel missense mutations in the antithrombin III (AT3) gene causing recurrent venous thrombosis

The polymerase chain reaction and direct sequencing were used to determine the nature of the mutations in the antithrombin III (AT3) gene in seven unrelated patients with familial antithrombin III (ATIII) deficiency and recurrent venous thrombosis. Three novel mutations were found, two associated with a type I deficiency state (Pro80Thr and His120Tyr) manifesting reduced synthesis of ATIII. The other novel lesion (Met251Ile) was associated with a dysfunctional ATIII protein (type II ATIII deficiency) and is predicted to interfere either with a heparin-induced conformational change in the ATIII molecule or with docking to thrombin. A novel polymorphism (Tyr158Cys) was also found to occur in several individuals of Scandinavian origin.     

Candidate-gene exclusion in a family with inherited non-syndromic dental disorders

Objectives

Amelogenesis imperfecta, dentinogenesis imperfecta, and dentin dysplasia are the most common non-syndromic dental disorders. In this study, we analysed and localised the gene(s) responsible for inherited non-syndromic dental disorders in a Chinese family.

Methods

This study identified and researched non-syndromic dental disorders in a four-generation Chinese family, including four affected individuals whose clinical phenotype was atypical. Linkage analysis with seven polymorphic markers that localise to six different autochromosomes showed that the family was linked through chromosome 4q. All exons and exon–intron boundaries of dentin sialophosphoprotein (DSPP), enamelin (ENAM), and ameloblastin (AMBN), which are located on chromosome 4q, were sequenced in nine of the family members.

Results

Direct DNA sequence analysis revealed the existence of a G to A transversion in exon 4 (g.13081786G > A, c.727G > A, p.Asp243Asn, based on reference sequences NM_014208.3) of the DSPP gene, and this sequence variation correlated exactly with the presence of the disease.

Conclusion

These results indicate that mutation p.Asp243Asn is a highly probable cause of non-syndromic dental disorder in this Chinese family. The presence of symptom heterogeneity is possible, as the clinical classification system is hampered by the lack of close correlation between the subtype and the molecular defect.     

Deletion of dentin matrix protein-1 leads to a partial failure of maturation of predentin into dentin, hypomineralization, and expanded cavities of pulp and root canal during postnatal tooth development

The dentin matrix protein-1 (DMP-1) gene is identified in odontoblasts during both embryonic and postnatal development. In vitro study suggests that this noncollagen acidic phosphoprotein plays a role in mineralization. However, deletion of the Dmp-1 gene has little effect on tooth development during embryogenesis. To address the role of DMP-1 in tooth during postnatal development, we analyzed changes of dentinogenesis in Dmp-1 null mice from 3 days after birth to 1 year. Here we show that Dmp-1 null mice postnatally develop a profound tooth phenotype characterized by a partial failure of maturation of predentin into dentin, enlarged pulp chambers, increased width of predentin zone with reduced dentin wall, and hypomineralization. The tooth phenotype of these mice is strikingly similar to that in dentin sialophosphoprotein (Dspp) null mice and shares some features of the human disease dentinogenesis imperfecta III. We have also demonstrated that DSPP levels are reduced in Dmp-1 null mice, suggesting that DSPP is probably regulated by DMP-1 during dentinogenesis. Finally, we show the absence or delayed development of the third molar in Dmp-1 null mice, which is probably secondary to defects in Dmp-1 null bone. Taken together, these studies suggest that DMP-1 is essential for later dentinogenesis during postnatal development.     

Novel promoter and splice junction defects add to the genetic,clinical or geographic heterogeneity of β-thalassaemia in the Portuguese population

Summary In order to delineate the spectrum and the relative abundance of -globin gene defects causing thalassaemia in the Portuguese population, a representative sample was analysed including 51 -thalassaemia carriers along with 26 patients representing different clinical phenotypes. Seven mutations were identified, four of which [codon 39 (CT), 39%; intervening sequence (IVS)1 nucleotide (nt) 1 (GA), 26%; IVS1 nt 110 (GA), 17%; IVS1 nt6 (TC), 15%] account for 97% of 93 -thalassaemia chromosomes. Two previously undescribed mutations, namely a CT substitution at position — 90 in the proximal CACCC box, and the deletion of nucleotides 4 and 5 (AG) in IVS 2 were identified. The uncommon, though ubiquitous, GT transversion at codon 121 was found once upon haplotype V. Direct prenatal diagnosis can be offered to 95% of couples at risk of bearing a thalassaemic child.     

Absorbance difference spectra of the S-state transitions in Photosystem II core particles

Redox changes of the oxygen evolving complex in PS II core particles were investigated by absorbance difference spectroscopy in the UV-region. The oscillation of the absorbance changes induced by a series of saturating flashes could not be explained by the minimal Kok model (Kok et al. 1970) consisting of a 4-step redox cycle, S₀ S₁ S₂ S₃ S₀, although the values of most of the relevant parameters had been determined experimentally. Additional assumptions which allow a consistent fit of all data are a slow equilibration of the S₃ state with an inactive state, perhaps related to Ca²⁺-release, and a low quantum efficiency for the first turnover after dark-adaptation. Difference spectra of the successive S-state transitions were determined. At wavelengths above 370 nm, they were very different due to the different contribution of a Chl bandshift in each spectrum. At shorter wavelengths, the S₁ S₂ transition showed a difference spectrum similar to that reported by Dekker et al. 1984b and attributed to an Mn(III) to Mn(IV) oxidation. The spectrum of absorbance changes associated with the S₂ S₃ transition was similar to that reported by Lavergne 1991 for PS II membranes. The S₀ S₁ transition was associated with a smaller but still substantial absorbance increase in the UV. Differences with the spectra reported by Lavergne 1991 are attributed to electrostatic effects on electron transfer at the acceptor side associated with the S-state dependence of proton release in PS II membranes.Abbreviations Bis-Tris (bis[2-hydroxyethyl]imino-tris[hydroxymethyl]methane) - DCBQ 2,5-dichloro-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PS II Photosystem II - Q_A secondary electron acceptor of PS II - S₀ to S₄ redox state of the oxygen evolving complex - Z secondary electron donor of PS II     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

Molecular basis of glucose-6-phosphate dehydrogenase (G6PD) deficiency in three Taiwan aboriginal tribes

We have investigated glucose-6-phosphate dehydrogenase (G6PD) deficiency in 220 unrelated aboriginal male subjects who belong to three different tribes (Saisiat, Ami, and Yami) in Taiwan. Our results show that the G6PD deficiency rates for Saisiat, Ami, and Yami people are 9.0% (6/67), 6.1% (6/99), and 0% (0/54), respectively. Among these deficiency cases, 4 of 6 (66.7%) Saisiat subjects have the 493 AG mutation and one carries the 1376 GT mutation, whereas, in Ami subjects, we found that four of six (66.7%) affected males have the 592 CT mutation and one carries the 493 AG mutation. These results contrast with our previous findings for Taiwan Chinese, in whom the 1376 GT mutation is the major mutant allele and accounts for 52.3% of the deficiency cases. This is the first report of G6PD deficiency characterized at the DNA level in Taiwan aboriginal populations.     

Dentin phosphoprotein gene locus is not associated with dentinogenesis imperfecta types II and III.

Dentinogenesis imperfecta (DGI) is an autosomal dominant inherited dental disease which affects dentin production and mineralization. Genetic linkage studies have been performed on several multigeneration informative kindreds. These studies determined linkage between DGI type II and III and group-specific component (vitamin D-binding protein). This gene locus has been localized to the long arm of human chromosome 4 in the region 4q11-q21. Although this disease has been mapped to chromosome 4, the defective gene product is yet to be determined. Biochemical studies have suggested abnormal levels of dentin phosphoprotein (DPP) associated with DGI type II. This highly acidic protein is the major noncollagenous component of dentin, being solely expressed by the ectomesenchymal derived odontoblast cells of the tooth. The purpose of the present study was to establish whether DPP is associated with DGI types II and III, by using molecular biology techniques. The strategy was to use a synthetic degenerative DPP oligonucleotide probe to map this sequence to the long arm of human chromosome 4, 4q13-q21, by using somatic cell hybrids. Our results indicated that DPP is not localized to any region of human chromosome 4, thus suggesting that the DPP gene is not directly associated with DGI type II or DGI type III. Our data do not exclude the possibility that other proteins associated with DPP posttranslational modifications might be responsible for this genetic disease.     

Acclimation of barley to changes in light intensity: chlorophyll organization

Barley seedlings (Hordeum vulgare L. cv. Boone) were grown at 20°C with a 16h/8h light/dark cycle of either high (H) intensity (550 mole m^-2 s^-1) or low (L) intensity (55 mole m^-2 s^-1) white light. Plants were transferred from high to low (H L) or low to high (L H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod and a 3 cm apical segment removed for analysis. H control plants had greater chlorophyll (Chl) per leaf area and higher Chl a/b ratios than L controls. Analysis of Chl-protein complexes revealed that H and L plants had the same percentage of total Chl (62–65%) associated with Photosystem II (PS II), but that the organization of Chl within PS II was different. H plants contained lower levels of light-harvesting complex (LHC-II) and higher levels of the PS II complex CPa compared with L plants. Leaf Chl content and Chl organization within PS II were sensitive to changes in light intensity. In H L plants, leaf Chl content decreased, Chl a/b ratio decreased, and a redistribution of Chl from CPa to LHC-II occurred during acclimation to low light. Acclimation of L H plants to high light involved an increase in leaf Chl content, an increase in Chl a/b ratio, and a decrease in LHC-II. In contrast, the level of photosystem I related Chl-protein complexes (CP1 + CP1a) was similar in all light treatments. The light acclimation process occurred slowly over a period of 6 to 8 d in H L and L H plants.Abbreviations DMF dimethylformamide - H control plants grown under high light intensity - H L plants transferred from high to low light intensity - L control plants grown under low light intensity - L H plants transferred from low to high light intensity Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 11989 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.