Characterization of a novel dentin matrix acidic phosphoprotein. Implications for induction of biomineralization

Acidic phosphorylated proteins have been shown to be prominent constituents of the extracellular matrix of bone and dentin. The acidic phosphoproteins of bone contain more glutamic acid than aspartic acid and a lower serine content than either. On the other hand, the major dentin acidic phosphoproteins, phosphophoryns, have been defined as aspartic acid- and serine-rich proteins, with a lesser content of glutamic acid. Both sets of phosphoproteins have been implicated as key participants in regulating mineralization, but it has been difficult to unify their mechanisms of action. We have now identified, by cDNA cloning, a new serine-rich acidic protein of the dentin matrix, AG1, with a composition intermediate between the bone acidic proteins and dentin phosphophoryns. AG1 has numerous acidic consensus sites for phosphorylation by both casein kinases I and II. Immunochemical and organ culture biosynthetic studies show that AG1 is present in phosphorylated form at low levels in the dentin matrix. If fully phosphorylated, AG1 would bear a net charge of -175/molecule of 473 residues. AG1 contains single RGD integrin binding and N-glycosylation sequences. The overall picture that emerges is that of a matrix-associated acidic phosphoprotein, with a potentially high calcium ion binding capacity, present at levels compatible with a regulatory role in dentin mineralization.     

A novel organ culture method to study the function of human odontoblasts in vitro: gelatinase expression by odontoblasts is differentially regulated by TGF-beta1

Odontoblasts cannot be cultured by traditional cell culture methods, thus restricting in vitro studies. Here we present an organ culture method for human odonto-blasts that utilizes the pulp chamber as a culture crucible. Crowns of human third molars were dissected, pulp was gently removed, and the odontoblasts attached to and in the walls of the pulp chambers were cultured in serum-free OPTI-MEM medium, or DMEM/Ham's F12 medium containing 10% serum. Pulp tissues were cultured separately. Cell content and morphology were analyzed by SEM, and the removed pulps were examined by light microscopy. Proteins secreted into the medium with or without TGF-beta1 supplementation were metabolically labeled with [35S]methionine, and the total protein content was assessed by TCA precipitation and SDS-PAGE/fluorography. To assess the role of gelatinolytic enzymes on dentin matrix remodeling, we used enzymography to analyze the effect of TGF-beta1 on gelatinase A and B expression. SEM revealed odontoblasts in pulp chambers after 5 days of culture, with only few or no fibroblasts, and no alterations in the odontoblast cell morphology or differences between the cells cultured in serum-free and serum-containing media. Rarely were any odontoblasts present in pulp tissue. Radiolabeling revealed protein synthesis and secretion until day 6 in both the odontoblast and pulp cultures, with no marked differences between TGF-beta1-treated and control cultures. The level of gelatinase A remained constant up to 7 days, while gelatinase B expression was always low and decreased with time in culture. However, gelatinase B levels were markedly increased upon TGF-beta1 treatment of cells and remained high to day 7. The results suggest that this method provides a novel technique for the study of human odontoblasts in vitro and that odontoblasts can be cultured even in serum-free conditions.     

Clinical features of five pedigrees genetically linked to the juvenile glaucoma locus on chromosome 1q21-q31

BACKGROUND: Primary juvenile glaucoma is a rare form of glaucoma that typically affects individuals between 3 and 20 years of ages and is inherited as an autosomal dominant trait. One gene responsible for this condition has been localized to the 1q21-q31 region of chromosome 1. To investigate the clinical features of this form of glaucoma, the authors have examined the affected members of five pedigrees demonstrating genetic linkage to the 1q21-q31 locus. METHODS: Clinical characterization of 23 affected patients was performed. Genetic linkage to the 1q21-q31 locus was confirmed by segregation of the disease trait in each pedigree with genetic markers located in the 1q21-q31 region. RESULTS: The clinical features of affected members of the five pedigrees presented are generally homogeneous. The average age of diagnosis was 18.5 years (range, 5-30 years), and the average initial intraocular pressure was 38.5 mmHg (range, 30-53 mmHg). Eighty-seven percent of affected individuals were myopic and 83% of affected individuals required surgical treatment for glaucoma. There were no uniformly associated systemic or ocular conditions. One possible nonpenetrant carrier was identified and a difference in phenotypic expression of the presumed disease gene was observed in a pair of affected monozygotic twins. We also identified two pedigrees with juvenile glaucoma and three pedigrees affected by the pigment dispersion syndrome that are not genetically linked to the 1q21-q31 region. CONCLUSION: The form of juvenile glaucoma caused by a gene located in the q21-q31 region of chromosome 1 is generally phenotypically homogeneous. The severe elevation of intraocular pressure typically seen in affected patients suggests the product of the predisposing gene may participate in the outflow function of the eye.     

Frequent microsatellite instability and loss of heterozygosity in the region including BRCA1 (17q21) in young patients with gastric cancer

It is known that nearly 5% of gastric carcinomas arise under the age of 40. To elucidate genetic alterations in these patients, we performed studies using microsatellite assay in 27 gastric cancers under 35 years of age, composed of 5 well and 22 poorly differentiated adenocarcinomas. We detected replication errors (RERs) in 18 (67%) of 27 tumors, but no germline mutation in DNA mismatch repair genes (hMLH1 and hMSH2), except fory 3 somatic mutations in the hMLH1 gene. Loss of heterozygosity (LOH) at D17S855, located on chromosome 17q21 (BRCA1), was detected in 8 (40%) of 20 informative cases. In 12 (44%) of 27 cases, LOH on chromosome 17q12-21 including the BRCA1 was found in several neighboring markers in this region, while no mutation was found in the BRCA1 gene. Four (40%) of 10 scirrhous type gastric cancers exhibited wide allelic deletions on chromosome 17q12-21. These results overall suggest that young gastric cancer patients display highly frequent micro-satellite instability that might be due to defect of DNA repair system rather than hMLH1 and hMSH2. In addition, chromosome 17q12-21 including BRCA1 locus may contain a candidate for tumor suppressor gene, particularly in scirrhous type gastric cancers arising in young patients.     

Refined chromosomal localization of the human thrombopoietin gene to 3q27-q28 and exclusion as the responsible gene for thrombocytosis in patients with rearrangements of 3q21 and 3q26

Thrombocytosis is a characteristic clinical feature in patients with myelocytic malignancies and chromosomal rearrangements of 3q21 and 3q26, sometimes called the '3q21q26 syndrome'. The function of thrombopoietin (TPO) in megakaryocytopoiesis and thrombopoiesis as well as its chromosomal location, marked TPO as a candidate gene for malignancies with 3q rearrangements combined with dysmegakaryopoiesis. In this study 12 cases with inv(3)(q21q26) or t(3;3)(q21;q26) were analyzed by means of PFGE, but no rearrangements near the TPO locus were detectable. Six YACs containing the TPO locus were isolated and characterized. By dual color in situ hybridization using a YAC from 3q26 containing the EVI1 gene and a YAC from the TPO locus, the localization of the human TPO gene could be refined to 3q27-q28 about 15-20 Mbp telomeric to the 3q26 breakpoints occurring in myeloid malignancies. TPO levels were analyzed in the serum of three patients and were found to be in the normal range. These results confirm the findings of two previous studies that thrombopoietin expression is not the main cause of thrombocytosis in the 3q21q26 syndrome.     

Identification of mutations in cystatin B, the gene responsible for the Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1)

Progressive myoclonus epilepsy (EPM1) is an autosomal recessive disorder, characterized by severe, stimulus-sensitive myoclonus and tonic-clonic seizures. The EPM1 locus was mapped to within 0.3 cM from PFKL in chromosome 21q22.3. The gene for the proteinase inhibitor cystatin B was recently localized in the EPM1 critical region, and mutations were identified in two EPM1 families. We have identified six nucleotide changes in the cystatin B gene of non-Finnish EPM1 families from northern Africa and Europe. The 426G-->C change in exon 1 results in a Gly4Arg substitution and is the first missense mutation described that is associated with EPM1. Molecular modeling predicts that this substitution severely affects the contact of cystatin B with papain. Mutations in the invariant AG dinucleotides of the acceptor sites of introns 1 and 2 probably result in abnormal splicing. A deletion of two nucleotides in exon 3 produces a frameshift and truncates the protein. Therefore, these four mutations are all predicted to impair the production of functional protein. These mutations were found in 7 of the 29 unrelated EPM1 patients analyzed, in homozygosity in 1, and in heterozygosity in the others. The remaining two sequence changes, 431G-->T and 2575A-->G, probably represent polymorphic variants. In addition, a tandem repeat in the 5' UTR (CCCCGCCCCGCG) is present two or three times in normal alleles. It is peculiar that in the majority of patients no mutations exist within the exons and splice sites of the cystatin B gene.     

A disease locus for familial hypertrophic cardiomyopathy maps to chromosome 1q3

Familial hypertrophic cardiomyopathy (FHC) is caused by missense mutations in the beta cardiac myosin heavy chain (MHC) gene in less than half of affected individuals. To identify the location of another gene involved in this disorder, a large family with FHC not linked to the beta MHC gene was studied. Linkage was detected between the disease in this family and a locus on chromosome 1q3 (maximum multipoint lod score = 8.47). Analyses in other families with FHC not linked to the beta MHC gene, revealed linkage to the chromosome 1 locus in two and excluded linkage in six. Thus mutations in at least three genetic loci can cause FHC. Three sarcomeric contractile proteins--troponin I, tropomyosin and actin--are strong candidate FHC genes at the chromosome 1 locus.     

The camptodactyly-arthropathy-coxa vara-pericarditis syndrome: clinical features and genetic mapping to human chromosome 1

OBJECTIVE: To delineate the clinical features in patients with the autosomal recessive camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) and to determine the location of the involved gene. METHODS: Eight affected individuals (ages 2-15 years) with CACP from 4 consanguineous kindreds were clinically evaluated. Four patients are newly described and 4 have been reported previously. Findings were compared with those in 21 other previously reported cases. DNA obtained from the 8 affected patients and their available siblings and parents was used in a genome-wide search for linkage. RESULTS: Congenital camptodactyly and childhood-onset noninflammatory arthropathy were present in all affected patients. Seven patients developed bilateral coxa vara deformity, and 1 developed coxa magna with cystic erosions. Two of the patients also had symptoms or signs of pericarditis. A genome-wide search for linkage identified homozygosity for a series of genetic markers on human chromosome 1q in all affected patients. The marker D1S191 yielded a maximum logarithm of the odds ratio (LOD score) of 3.3 at theta = 0. The CACP gene lies within a 1.9-cM candidate interval defined by the markers D1S2107 and D1S222. CONCLUSION: The principal features of the CACP syndrome are congenital or early-onset camptodactyly and childhood-onset noninflammatory arthropathy. Coxa vara deformity or other dysplasia associated with progressive hip disease may develop over time. Clinical pericarditis may also occur. A locus responsible for causing CACP syndrome is assigned to a 1.9-cM interval on human chromosome 1q25-31 by homozygosity mapping. This now facilitates the identification of the responsible gene and permits testing for locus homogeneity in other CACP kindreds.     

Mapping of the gene encoding the B56 beta subunit of protein phosphatase 2A (PPP2R5B) to a 0.5-Mb region of chromosome 11q13 and its exclusion as a candidate gene for multiple endocrine neoplasia type 1 (MEN1)

The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localised to 11q13 by combined tumour deletion mapping and recombination studies, and a 0.5-Mb region, flanked by PYGM and D11S449, has been defined. In the course of constructing a conting, we have identified the location of the gene encoding the B56 beta subunit of protein phosphatase 2A (PP2A), which is involved in cell signal transduction pathways and thus represents a candidate gene for MEN1. We have searched for mutations in the PP2A-B56 beta coding region, together with the 5' and 3' untranslated regions in six MEN1 patients. DNA sequence abnormalities were not identified and thus the PP2A-B56 beta gene is excluded as the candidate gene for MEN1. However, our precise localisation of PP2A-B56 beta to this region of 11q13 may help in elucidating the basis for other disease genes mapping to this generich region.     

Expression and localization of membrane type 1 matrix metalloproteinase in tooth tissues

Matrix metalloproteinases (MMPs) have been detected in forming dental enamel and are thought to play an important role during enamel biomineralization. Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane bound member of the MMP gene family that has previously been shown to be expressed by cells associated with bone and cartilage formation (osteoclasts, osteoblasts and chondrocytes). Thus, we asked if MT1-MMP was also expressed by the cells responsible for the formation of enamel and dentin. A porcine MT1-MMP cDNA composed of 3284 bp was isolated from an enamel organ-specific cDNA library. Multiple tissue Northern blot analysis revealed that the MT1-MMP message was expressed highly in the enamel organ and pulp organ when compared to the expression levels observed in other non-mineralizing tissues. Northern blot analysis of stage-specific enamel organs (early secretory, late secretory, or maturation stage) and their corresponding pulp organs revealed that MT1-MMP expression increased as the dentin matured. In the enamel organs, however, the MT1-MMP message level became reduced only during the late secretory stage. Immunohistochemical analysis showed that MT1-MMP was present on the surface of the cells (ameloblasts and odontoblasts) responsible for dentin and enamel formation. Thus, MT1-MMP is highly expressed in developing tooth tissues and may play a role in the biomineralization of enamel and dentin.     

Assessment of the interphotoreceptor matrix proteoglycan-1 (IMPG1) gene localised to 6q13-q15 in autosomal dominant Stargardt-like disease (ADSTGD), progressive bifocal chorioretinal atrophy (PBCRA), and North Carolina macular dystrophy (MCDR1)

We have recently characterised the genomic organisation of a novel interphotoreceptor matrix proteoglycan, IMPG1, and have mapped the gene locus to chromosome 6q13-q15 by fluorescence in situ hybridisation. As the interphotoreceptor matrix (IPM) is thought to play a critical role in retinal adhesion and the maintenance of photoreceptor cells, it is conceivable that a defect in one of the IPM components may cause degenerative lesions in retinal structures and thus may be associated with human retinopathies. By genetic linkage analysis, several retinal dystrophies including one form of autosomal dominant Stargardt-like macular dystrophy (STGD3), progressive bifocal chorioretinal atrophy (PBCRA), and North Carolina macular dystrophy (MCDR1) have previously been localised to a region on proximal 6q that overlaps the IMPG1 locus. We have therefore assessed the entire coding region of IMPG1 by exon amplification and subsequent single stranded conformational analysis in patients from 6q linked multigeneration families diagnosed with PBCRA and MCDR1, as well as a single patient from an autosomal dominant STGD pedigree unlinked to either of the two known STGD2 and STGD3 loci on chromosomes 13q and 6q, respectively. No disease associated mutations were identified. In addition, using an intragenic polymorphism, IMPG1 was excluded by genetic recombination from both the PBCRA and the MCDR1 loci. However, as the autosomal dominant Stargardt-like macular dystrophies are genetically heterogeneous, other forms of this disorder, in particular STGD3 previously linked to 6q, may be caused by mutations in IMPG1.     

Autosomal dominant medullary cystic kidney disease: evidence of gene locus heterogeneity

Autosomal dominant medullary cystic kidney disease (ADMCKD; synonym: medullary cystic disease, MCD) is an autosomal dominant kidney disorder, sharing morphological and clinical features with recessive juvenile nephronophthisis (NPH), such as reduced urinary concentration ability and multiple renal cysts at the corticomedullary junction. While in NPH end-stage renal disease (ESRD) occurs in adolescence, ADMCKD leads to ESRD in adulthood. Recently a gene locus for ADMCKD has been localized to chromosome 1q21 in two large Cypriot families. This prompted us to examine linkage in three ADMCKD-families, using the same set of polymorphic microsatellite markers spanning the critical region on chromosome 1q21. Haplotype analysis revealed that none of the three families showed linkage to this locus, thus demonstrating evidence for genetic locus heterogeneity. Additional linkage analysis studies need to be performed in order to identify further gene loci cosegregating with this autosomal dominant kidney disorder.     

A novel Asp380Ala mutation in the GLC1A/myocilin gene in a family with juvenile onset primary open angle glaucoma

Glaucoma describes a clinically and genetically heterogeneous group of diseases that result in optic neuropathy and progressive loss of visual fields. A gene for juvenile onset primary open angle glaucoma JOAG) has recently been mapped to 1q21-31. Mutations in the trabecular meshwork induced glucocorticoid response gene (TIGR, also known as myocilin or the GLC1A locus) have been found to cause both juvenile and later onset primary open angle glaucoma. Family TCD-POAG1 is a Spanish kindred, which segregates JOAG in an autosomal dominant fashion. This family was found to be linked to the previously identified GLC1A locus on chromosome 1q. Direct sequencing of the TIGR/myocilin gene showed a heterozygous A to C transition in codon 380, resulting in the substitution of alanine for aspartic acid (Asp380Ala). This substitution created a StyI restriction site, which segregated with the JOAG phenotype and permitted rapid screening of all members of the family. This restriction site was not present in 60 controls.