Relationships between bone mass and dietary/lifestyle habits in Japanese women at 3-4 months postpartum.

OBJECTIVES: The relationships between calcaneal bone mass and dietary/lifestyle habits in women at 3-4 months postpartum were examined in the context of osteoporosis prevention. STUDY DESIGN: Cross-sectional survey. METHODS: We measured bone mass using calcaneal ultrasound in mothers who brought their 3- to 4-month-old babies to healthcare centers in Japan for health examination and administered a self-report questionnaire on physical characteristics and dietary/lifestyle habits to those who agreed to participate in the survey. Valid data were available for 1220 women (valid response rate, 97.5%). RESULTS: Based on their stiffness score, a measure of bone mass, 70.9% (n = 865) of the participants were classified as 'no apparent abnormality (stiffness score ≥78.8)' (low-risk group), 18.2% (n = 222) as 'guidance required (≥70.1-<78.8)' (intermediate-risk group), and 10.9% (n = 133) as 'complete examination required (<70.1)' (high-risk group), according to the criteria for osteoporosis screening test results. The percentage of individuals with a history of fracture was higher in the guidance required/complete examination required than in the no apparent abnormality group (P = 0.016). The analysis of relationships between the consumption frequency of certain foods, such as calcium-rich foodstuffs, and bone mass found that women who reported lower frequencies of milk and dark-colored (beta-carotene rich) vegetables for breakfast consumption had a significantly lower bone mass than those who consumed these foods more often. Furthermore, the guidance required/complete examination required group had a significantly lower calcium intake than the no apparent abnormality group (P = 0.022). CONCLUSIONS: These results indicate the need to provide postpartum women with dietary education programs to promote healthy eating habits, such as increased consumption of calcium-rich foods, and prevent osteoporosis.

Identification of a novel frameshift mutation (383insT) in the RUNX2 (PEBP2 alpha/CBFA1/AML3) gene in a Japanese patient with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant disorder due to mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer-binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1)/acute myeloid leukemia 3 (AML3). To investigate the RUNX2 mutations in a Japanese patient with classic CCD, we analyzed the RUNX2 gene using polymerase chain reaction (PCR)-single-strand conformation polymorphism and PCR-restriction fragment length polymorphism. The patient had hypoplasia of the clavicles, patent fontanelles, short stature, supernumerary teeth, and retention of deciduous dentition. We identified a 1-bp insertion (383insT) at codon 128 of the RUNX2 gene. The 383T insertion affects the conserved residue in the runt domain and results in premature termination in the runt domain.

Mutational analysis and functional correlation with phenotype in German patients with childhood-type hypophosphatasia.

The tissue-nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood-type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP)-direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS-1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80-kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high-molecular mass disulfide-linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood-type HOPS.

A novel point mutation (C571T) in the tissue-non-specific alkaline phosphatase gene in a case of adult-type hypophosphatasia.

OBJECTIVE: Hypophosphatasia (HOPS) is an inheritable disorder characterized by defective skeletal mineralization, deficiency of tissue-non-specific alkaline phosphatase (TNSALP) activity and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34-36.1 and consists of 12 exons and 11 introns. In this study we analysed the genomic TNSALP gene from a patient with HOPS, her family, and unrelated normal controls. MATERIALS AND METHODS: The proband was a 52-year-old Japanese woman with adult onset HOPS. The patient showed deficiency in alkaline phosphatase (ALP) activity, increased urinary excretion of phosphoethanolamine and severe periodontal disease. Genomic DNA was extracted from the peripheral leukocytes of the subjects. Based on published sequence data in the TNSALP gene, 11 pairs of polymerase chain reaction (PCR) primers were used to amplify the coding exons. The PCR amplified samples were subjected to PCR-single strand conformation polymorphism (SSCP) analysis and PCR-allele specific oligonucleotide (ASO) analysis. RESULTS: By PCR-SSCP analysis of the patient's genomic DNA, fragments containing exon 5 revealed abnormal mobility. This abnormal mobility (exon 5) was also found in the genomic DNA in her mother's sister, but were not detected in her father, brothers or sisters, and unrelated normal controls. Sequencing analysis of the abnormal band extracted from the SSCP gel revealed a C to T transition at nucleotide position 571 (C571T) in exon 5. This mutation resulted in a substitution of Ala-115 with a Val in the mature TNSALP polypeptide. PCR-ASO analysis also confirmed this missense point mutation. The result of this study showed that the pro-band has inherited the C571T mutation in exon 5 from her mother alone and the disease in this family was inherited as an autosomal dominant trait from the pedigree. CONCLUSIONS: The C571T mutation is a new missense point mutation and appears to cause significant changes in the structure and function of TNSALP because Ala-115 is highly conserved in rat TNSALP and human tissue-non-specific, intestinal and placental ALPs.

A case of Japanese cleidocranial dysplasia with a CBFA1 frameshift mutation.

Cleidocranial dysplasia (CCD), which is caused by mutations of the core binding factor alpha 1 (CBFA1)/runt-related gene 2 (Runx2), is an autosomal, dominantly inherited disorder of high penetrance affecting skeletal ossification and tooth development. Recently, we found a novel frameshift mutation 383-T-insertion (S128F) in exon 3 in the CBFA1 gene of a Japanese classic CCD patient. We describe our detailed investigation of the patient with CCD associated with the CBFA1 mutation. The patient showed the characteristic expression of CCD, such as dysplasia of the clavicles, patent fontanelles, short stature, impacted supernumerary teeth, and delayed eruption of the permanent teeth. In addition to these characteristics, orthopantomography delayed ossification of the mandibular symphysis and a three-dimensional computed tomograph (3D-CT) analysis showed hypoplasia of the zygomatic arch. Furthermore, the acellular cementum of an impacted supernumerary tooth was absent in this patient. Thus, the CBFA1 mutation was critical for the pathogenesis of CCD in this patient.

Phosphate depletion enhances tissue-nonspecific alkaline phosphatase gene expression in a cultured mouse marrow stromal cell line ST2.

Alkaline phosphatases (ALP) are highly ubiquitous enzymes present in the majority of animals from bacteria to higher vertebrate. Although their wide distribution in nature has suggested that these enzymes should perform important biological functions, their detailed roles or natural substrates remain unknown. In Escherichia coli, the extracellular phosphate (Pi) limitation induces the ALP gene, indicating the role of extracellular Pi in ALP gene regulation. However, little is known about the similar mechanisms in mammalian cells. This study was designed to examine the effect of low Pi medium on the ALP activity and its expression in the mouse stromal cell line ST2. The enzymatic property was classified into tissue-nonspecific ALP (TNSALP). After treatment by Pi starvation for 3 days, there was a 2-fold increase in the specific activity of TNSALP. RT-PCR analysis revealed that the mRNA of the TNSALP gene was highly stimulated. These results indicated that the effect of Pi depletion on ALP activity was regulated at the TNSALP transcriptional level, suggesting that the possible role of the Pi sensing system for biological functions of ALP might have been conserved in evolution. Our findings also made it possible to discuss the physiological roles of ALP in vivo.

Tissue-non-specific alkaline phosphatase mRNA expression and alkaline phosphatase activity following application of retinoic acid in cultured human dental pulp cells.

Retinoic acid is a potent inducer of tissue-non-specific alkaline phosphatase (TNSALP) expression in various osteoblastic and fibroblastic cells, and may be involved in morphogenesis, cellular growth and differentiation. This study investigates the effects of retinoic acid on alkaline phosphatase activity and TNSALP gene expression in human dental pulp cells. Cultured cells were treated with various concentrations of retinoic acid (0, 10(-7), 10(- 6), 10 (-5) M) in 0.5% bovine serum albumin without serum. Alkaline phosphatase activity was determined by the rate of p-nitrophenyl phosphate hydrolysis and was also assayed in the presence of various inhibitors and under thermal inactivation. A set of specific oligonucleotide primers was selected, based on the nucleotide sequences of two human TNSALP mRNA (bone and liver) types, and reverse transcription-polymerase chain reaction (RT-PCR) performed. Inhibitory and thermal inactivation experiments revealed that the elevated alkaline phosphatase activity had properties of the TNSALP type. RT-PCR showed that retinoic acid enhanced the expression of bone-type TNSALP mRNA in pulp cells. However, the liver-type TNSALP mRNA was not detected. These findings suggest that the high alkaline phosphatase activity of retinoic acid-treated dental pulp cells is associated with increased transcription of the bone-type mRNA of the TNSALP gene and not with liver-type.

Molecular diagnosis of hypophosphatasia with severe periodontitis.

Hypophosphatasia (HOPS) is an inherited disorder characterized by the defect of skeletal mineralization due to tissue-nonspecific alkaline phosphatase (TNSALP) deficiency. In this study we analyzed the TNSALP gene from a Japanese patient with HOPS, his parents, his brother, and unrelated normal controls. The proband is a 25-year-old Japanese male diagnosed with childhood hypophosphatasia. The patient reported premature exfoliation of the deciduous teeth and severe periodontal destruction of the permanent dentition. Genomic DNA was extracted from peripheral leukocytes of subjects. Eleven pairs of the polymerase chain reaction (PCR) primers were used to amplify the coding exons according to the published sequence data of the TNSALP gene. The PCR amplified samples were subjected to PCR-single strand conformation polymorphism (SSCP) analysis and PCR-allele specific oligonucleotide (ASO) analysis. In PCR-SSCP analysis of the patient's genomic DNA, the fragments containing exons 9 and 10 revealed abnormal mobilities. These abnormal mobilities (exons 9 and 10) were also found from his mother and father's genomic DNA, respectively. The sequencing analysis of the abnormal bands extracted from the SSCP gel showed a T to C transition at nucleotide position 1155 (T1155C) in exon 9 and G1320A in exon 10. PCR-ASO analysis confirmed these missense point mutations. PCR-ASO analysis also confirmed that mutation-specific oligonucleotides corresponded to the new mutations and did not hybridize with PCR products from normal control genomic DNAs. These results indicated that the proband was a compound heterozygote who inherited T1155C mutation in exon 9 from the mother and G1320A mutation in exon 10 from the father. Both of them are new missense point mutations and appear to cause significant changes in the structure and function of TNSALP.

Expression of mRNA encoding tissue-nonspecific alkaline phosphatase in human dental tissues.

Tissue-nonspecific-type alkaline phosphatase (TNSALP) is found in the bone, liver, kidney, and other tissues, and its gene consists of 12 exons with the coding sequence beginning in the second exon. Recently, a noncoding first exon was identified in the liver message (liver type) which differed from that of the previously known osteoblast-derived cDNA sequence (bone type). Although these two mRNAs produce an identical protein, they have different promoter regions. It is known that ALPs in dental pulp and periodontal ligament are classified into TNSALP by their enzymatic and immunological properties, but little is known about the expression of ALP mRNAs and the transcriptional mechanisms. In order to examine the expression of their mRNA type, specific oligonucleotide primers corresponding to two types of mRNAs of human TNSALP were designed and amplified by reverse transcription-polymerase chain reaction (RT-PCR). It was found that bone-type mRNA was expressed in the human dental tissues such as dental pulp, periodontal ligament, and dental sac, whereas liver-type mRNA was not expressed. Thus, it was concluded that the human dental tissues express the bone-type isozymes and are regulated by the same transcriptional mechanism as in the bone.

The effects of retinoic acid on alkaline phosphatase activity and tissue-non-specific alkaline phosphatase gene expression in human periodontal ligament cells and gingival fibroblasts.

Alkaline phosphatase (ALP) in human periodontal ligament (HPDL) cells is classified as a tissue-non-specific alkaline phosphatase (TNSALP) by its enzymatic and immunological properties. Since retinoic acid (RA) has been shown as a potent inducer of TNSALP expression in various osteoblastic and fibroblastic cells, we investigated the effects of RA on the level of ALP activity and expression of TNSALP mRNAs in HPDL cells. Cultured cells were treated with desired RA concentrations (0, 10(-7), 10(-6), 10(-5) M) in medium containing 1% bovine serum albumin without serum. ALP activity was determined by the rate of hydrolysis of p-nitrophenyl phosphate and was also assayed in the presence of specific inhibitors. In order to identify the TNSALP mRNA type expressed by HPDL, a set of oligonucleotide primers corresponding to 2 types of human TNSALP mRNA (i.e. bone-type and liver-type) were designed, and mRNA isolated from HPDL was amplified by means of reverse transcription-polymerase chain reaction (RT-PCR). After treatment with RA (10(-6) M) for 4 d, there was a significant increase in the ALP activity of HPDL cells. The use of inhibitors and thermal inactivation experiments showed that the increased ALP activity had properties of the TNSALP type. RT-PCR analysis revealed that bone-type mRNA was highly stimulated in HPDL cells by RA treatment, but the expression of liver-type mRNA was not detected. These results indicated that the upregulation of ALP activity in HPDL cells by RA was due to the increased transcription of bone-type mRNA of the TNSALP gene.

Expression of the mutant (1735T-DEL) tissue-nonspecific alkaline phosphatase gene from hypophosphatasia patients.

Hypophosphatasia (HOPS) is an inherited disorder characterized by defects in skeletal mineralization due to the deficiency of tissue-nonspecific alkaline phosphatase (TNSALP). To date, various mutations in the TNSALP gene have been identified. Especially, a deletion of T at position 1735 (1735T-del) located in exon 12 has been detected in three genetically unrelated Japanese patients, which seems to be one of the hot spots among the causative mutations in Japanese HOPS patients. 1735T-del causes a frame shift downstream from codon 503 (Leu), and consequently the normal termination codon at 508 is eliminated. Since a new inframe termination codon appears at codon 588 in the mutant DNA, the resultant protein is expected to have 80 additional amino acids. Expression of the mutant TNSALP gene using COS-1 cells demonstrated that the protein translated from the mutant 1735T-del had undetectable ALP activity, and its molecule size was larger than normal, as expected. Interestingly, an immunoprecipitation study of patients' sera using antibody against TNSALP revealed an abnormal protein which corresponded in size to the mutated TNSALP expressed by COS-1 cells, suggesting that the abnormal TNSALP is made by HOPS patients. The detection of TNSALP in cells transfected with 1735T-del using an immunofluorescent method exhibited only a faint signal on the cell surface, but an intense intracellular fluorescence after permeabilization.

Gene expression of growth and differentiation factors-5, -6, and -7 in developing bovine tooth at the root forming stage.

Growth and differentiation factors (GDF)-5, -6, and -7 are members of the bone morphogenetic protein (BMP) family. Previous studies suggest their importance in bone development and in tendon/ligament morphogenesis. The cells of the dental attachment apparatus, cementum, periodontal ligament, and alveolar bone proper are derived from the dental follicle proper. In this study, we investigated the expression of GDF-5, -6, and -7 genes in tissues of the bovine incisor tooth germ at the root forming stage. The results demonstrate distinct expression of GDFs in both the dental follicle and the odontoblast layer. While GDF-5 and -6 mRNAs were expressed in both the dental follicle and the odontoblast layer, GDF-7 mRNA expression was detected only in the dental follicle. These results indicate that GDFs, expressed in the bovine tooth germ including the dental follicle, may be potent regulatory molecules in the development of the dental attachment apparatus.

Effects of ovariectomy on intestinal alkaline phosphatase expression in rats.

The aim of this study was to investigate the effects of ovariectomy (OVX) on intestinal alkaline phosphatase (ALP) activity in rats. The calcium (Ca) and phosphorus (P) contents and the mechanical strength of bone were decreased significantly by OVX. Two kinds of mRNAs of rat intestinal ALP (RTIN-1 and RTIN-2) were detected by reverse transcription-polymerase chain reaction (RT-PCR). In OVX rats, the level of RTIN-2 mRNA was lowered significantly, while that of RTIN-1 mRNA did not change. This result was compatible with the results of enzymatic activity. This finding suggests the possibility that OVX affects bone metabolism not only directly but also in an indirect way through an intestinal Ca and/or P metabolism via regulation of intestinal RTIN-2 ALP expression.

Expression of mRNA encoding intestinal type alkaline phosphatase in rat liver and its increase by fat-feeding.

The presence of types of alkaline phosphatase (ALP) other than the tissue non-specific type enzyme in rat liver and its increase by fat feeding are known. In order to examine expression of intestinal type ALP in liver, specific oligonucleotide primers corresponding to two types of mRNAs of rat intestinal ALP (RTIN-1 and -2) were designed and amplified by means of the reverse transcriptase-polymerase chain reaction (RT-PCR). It was found that RTIN-1 mRNA was expressed only in the intestine but not in the liver, while RTIN-2 mRNA was expressed both in the intestine and in the liver. By fat feeding, expression of RTIN-1 mRNA increased in the intestine and that of RTIN-2 mRNA increased both in the intestine and in the liver. Thus, it was concluded that rat liver expressed one of the intestinal type ALP (RTIN-2) which was enhanced by fat feeding.