Genetic mapping of the dentinogenesis imperfecta type II locus.

Dentinogenesis imperfecta type II (DGI-II) is an autosomal dominant disorder of dentin formation, which has previously been mapped to chromosome 4q12-21. In the current study, six novel short tandem-repeat polymorphisms (STRPs) have been isolated, five of which show significant evidence of linkage to DGI-II. To determine the order of the STRPs and define the genetic distance between them, nine loci (including polymorphisms for two known genes) were mapped through the CEPH reference pedigrees. The resulting genetic map encompasses 16.3 cM on the sex-averaged map. To combine this map with a physical map of the region, all of the STRPs were mapped through a somatic cell hybrid panel. The most likely location for the DGI-II locus within the fixed marker map is in the D4S2691-D4S2692 interval of 6.6 cM. The presence of a marker that shows no recombination with the DGI-II phenotype between the flanking markers provides an important anchor point for the creation of physical continuity across the DGI-II candidate region.     

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.     

Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III

Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels.     

A novel splice acceptor mutation in the DSPP gene causing dentinogenesis imperfecta type II

The dentin sialophosphoprotein (DSPP) gene (4q21.3) encodes two major noncollagenous dentin matrix proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Defects in the human gene encoding DSPP cause inherited dentin defects, and these defects can be associated with bilateral progressive high-frequency sensorineural hearing loss. Clinically, five different patterns of inherited dentin defects are distinguished and are classified as dentinogenesis imperfecta (DGI) types I, II, and III, and dentin dysplasia types I and II. The genetic basis for this clinical heterogeneity is unknown. Among the 11 members recruited from the studied kindred, five were affected with autosomal dominant DGI type II. The mutation (g.1188CG, IVS2-3CG) lay in the third from the last nucleotide of intron 2 and changed its sequence from CAG to GAG. The mutation was correlated with the affection status and was absent in 104 unaffected individuals (208 alleles) with the same ethnic and geological background. The proband was in the primary dentition stage and presented with multiple pulp exposures. The occlusal surface of his dental enamel was generally abraded, and the dentin was heavily worn and uniformly shaded brown. The dental pulp chambers appeared originally to be within normal limits without any sign of obliteration, but over time (by age 4), the pulp chambers became partially or completely obliterated. The oldest affected member (age 59) showed mild hearing loss at high-frequency (8 kHz). Permanent dentition was severely affected in the adults, who had advanced dental attrition, premature loss of teeth, and extensive dental reconstruction.     

Baculovirus phosphoprotein pp31 is associated with virogenic stroma.

The PstI K fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) encodes a protein with a molecular weight of 31,000. To define the role of this protein (pp31) in virus infection further, it was overexpressed in bacteria and used to produce polyclonal antiserum. Radioimmunoprecipitation analysis indicated that pp31 was synthesized during both the early and late phases of virus infection, consistent with previous analyses indicating that the gene was regulated by tandem early and late promoters. Metabolic labeling of cells with carrier-free phosphate indicated that pp31 was phosphorylated. Biochemical fractionation experiments showed that pp31 was localized in the nucleus and that it was more stably associated with the nucleus at later times of infection. Immunoblot analysis of subnuclear fractions indicated that pp31 was associated predominantly with the chromatin and nuclear matrix fractions. Immunofluorescence experiments confirmed that the pp31 protein was localized in the nucleus. Nuclear staining was relatively uniform early but was more centrally nuclear later in infection. Immunoelectron microscopy indicated that the pp31 protein was a component of virogenic stroma. Southwestern (DNA-protein) blot analysis demonstrated that pp31 is a DNA-binding protein. These findings suggest a possible role for pp31 in the virus life cycle.     

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.     

Clinical,histopathologic, and genetic investigation in two large families with dentinogenesis imperfecta type II

Dentinogenesis imperfecta (DI) type II, an inherited disorder affecting dentin, has been linked to mutations in the dentin sialophosphoprotein (DSPP) gene on chromosome 4q21. The gene product is cleaved into two dentin-specific matrix proteins, dentin sialoprotein (DSP) and dentin phosphoprotein. The aim of this investigation was to study genotypes and phenotypes in two affected families with special reference to clinical, radiographic, and histopathologic manifestations. Seven affected members of Family A and five of Family B were documented clinically and radiographically; 14 and 10 teeth, respectively, were available for histopathologic investigation and prepared for ground sections, which were assessed semiquantitatively for dysplastic manifestations in the dentin according to the scoring system, dysplastic dentin score (DDS). Venous blood samples were collected from six affected and ten unaffected members of Family A, and from eight affected and six unaffected members of Family B. Genomic DNA was extracted and used for sequence analyses. The two families presented with different missense mutations. An Arg68Trp missense mutation in the DSP part of the gene was revealed in all six analyzed affected individuals in Family A. This mutation was not present in any of the ten healthy members. In Family B, an Ala15Val missense mutation involving the last residue of the signal peptide was found in all eight affected but in none of the six healthy members. The clinical and radiographic disturbances and DDS were more severe in Family B. The data indicate the presence of a genotype-phenotype correlation in DI type II.     

Dental findings in osteogenesis imperfecta: I. Occurrence and expression of type I dentinogenesis imperfecta

Sixty-eight patients with osteogenesis imperfecta (OI) classified according to Sillence were evaluated for dentinogenesis imperfecta (DI). Orthopantomograms of 51 of the 68 were examined. Type I DI was recognized in 22 patients from 16 families. DI was observed in 4/45 patients with type I OI, in one of two patients with type III, and in 13/16 patients with type IV OI. Four of the five patients with an unidentified type of OI had DI. The expression of type I DI was variable. Discoloration and pulpal obliteration were the major manifestations. Teeth from 14 patients from 12 families were studied histologically. Eight of the 14 patients were from six families who had clinical and/or radiographic evidence of DI. Irregularity of the dentin matrix and tubular pattern in the circumpulpal dentin and normal mantle dentin were observed. Interfamilial variability was greater than intrafamilial variability. The expression of DI was mild in one family with type I OI. There was no further relation between the type of OI and the severity of DI.     

MTHFR gene variant is not associated with diabetic nephropathy in Japanese.

Genetic predisposition has been implicated in diabetic nephropathy. The C677T variant of the MTHFR gene has been suggested to play a role in the development of not only vascular diseases but also diabetic microangiopathies. By using polymerase chain reaction-restriction length polymorphism (PCR-RFLP) method using Hinf I, we investigated whether this variant is associated with diabetic nephropathy in Japanese. By analysing 274 unrelated Japanese patients with type II DM with or without nephropathy, there was no significant difference in the genotype distribution of this variant. The distribution of the three genotypes were not different among patients with micro- or macroalbuminuria and those without nephropathy. Although previous reports suggest a role of this variant with diabetic microangiopathies, our observations suggest that this variant is does not play an important role in the pathogenesis of diabetic nephropathy in Japanese.     

Activated murine alpha-globin gene is not preferentially associated with the nuclear matrix

The association of the murine alpha-globin gene with the nuclear matrix was studied in three different states of the gene: inactive (EAT cells), potentially active (MEL cells) and active (induced MEL cells). When "native" nuclei were digested with DNase I it was found that the nuclear matrix was not enriched in alpha-globin DNA sequences in all three different types of cells. A nuclease-hypersensitive site in the 5'-flanking region of the alpha-globin gene was detected in the induced MEL-cells.     

The gene for the 10 kDa phosphoprotein of photosystem II is located in chloroplast DNA

Nucleotide sequencing of a region of wheat chloroplast DNA between the genes for the 47 kDa chlorophyll a-binding protein of photosystem II (psbB) and cytochrome b-563 (petB) has revealed an open reading frame of 73 codons. This open reading frame has been identified as the gene (psbH) for the 10 kDa phosphoprotein of photosystem II by comparison with the published N-terminal amino acid sequence and amino acid composition of the purified spinach protein. The predicted sequence of the protein shows some homology with the N-terminal region of the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII).     

Proximal spinal muscular atrophy (SMA) types II and III in the same sibship are not caused by different alleles at the SMA locus on 5q.

Proximal spinal muscular atrophy (SMA) is a group of progressive muscular diseases recently mapped to chromosome 5q. SMA is usually classified into types I-III, and there are cases of two types of SMA in the same sibship. Becker and others later proposed that these sibships might be due to the existence of several alleles at the same locus predisposing to the different forms of the disease. In a sample of four sibships in which both SMA type II and SMA type III occur, this hypothesis was clearly rejected for the SMA locus on 5q, by using information on the segregation of linked markers (P less than .001). Thus the difference between SMA type II and SMA type III is not due to different alleles at the SMA locus on 5q. This finding is suggestive of an involvement of other factors, genetic or environmental, in the determination of disease severity in SMA.     

An intron mutation in the human alpha 1(I) collagen gene alters the efficiency of pre-mRNA splicing and is associated with osteogenesis imperfecta type II

This study describes a homozygous, G----A transition at the moderately conserved +5 position within the splice donor site of intron 14 in the human alpha 1(I) collagen gene. The mutation reduced the efficiency of normal splice-site selection since the exon upstream of the mutation was spliced alternatively. Moreover, the extent of alternative splicing was sensitive to the temperature at which the mutant cells were grown, suggesting that the mutation directly affected spliceosome assembly. To achieve exon skipping, this effect must be propagated so as to disrupt the selection of a second splice site in the adjacent intron.