Association of NPP1 polymorphism with postoperative progression of ossification of the posterior longitudinal ligament in Chinese patients.

Ossification of the posterior longitudinal ligament (OPLL) is a condition of the spine that can cause paralysis by compressing the spinal cord. The aim of this study was to evaluate the possible role of nucleotide pyrophosphatase phosphodiesterase 1 gene (NPP1) polymorphism in the etiology and pathology of the OPLL in Chinese patients. DNA from patients with OPLL (N = 95) and without OPLL (N = 90) were genotyped for 4 NPP1 single-nucleotide polymorphisms (SNPs): A533C, C973T, IVS15-14T→C, and IVS20-11delT. An association study evaluated the relationship between specific SNP genotypes and susceptibility. We also evaluated whether genotypes of these SNPs were associated with disease severity and the probability of disease progression after surgery. The C973T and IVS15-14T SNPs were associated with the existence of the disease. The TT genotypes of C973T and IVS15→14T as well wild-type IVS20 (lack of deletion) were associated with more severe disease. Patients with the T deletion of IVS20 or the AA genotype of A533C had an approximately 3 times greater chance of not having than having disease progression after surgery. We concluded that the 4 SNPs analyzed appeared to have different effects on the etiology and pathology of OPLL. To our knowledge, this study is the first to investigate the relationship between these SNPs and disease progression after surgery. Our findings suggest that the presence of specific genotypes of the IVS20-11delT and A533C SNPs may predict disease outcome after surgical intervention.

A patient with hypophosphatemic rickets and ossification of posterior longitudinal ligament caused by a novel homozygous mutation in ENPP1 gene.

X-linked hypophosphatemic rickets/osteomalacia (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and autosomal recessive hypophosphatemic rickets/osteomalacia (ARHR1 or ARHR2) are hereditary fibroblast growth factor 23 (FGF23)-related hypophosphatemic rickets showing similar clinical features. We here show a patient with hypophosphatemic rickets and widespread ossification of posterior longitudinal ligament (OPLL). The proband is a 62-year-old female. Her parents are first cousins and showed no signs of rickets or osteomalacia. She showed hypophosphatemic rickets with elevated FGF23 level and had been clinically considered to be suffering from XLH. However, direct sequencing of all coding exons and exon-intron junctions of phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX), FGF23 and dentin matrix protein 1 (DMP1) genes, responsible genes for XLH, ADHR and ARHR1, respectively, showed no mutation. A novel homozygous splice donor site mutation was found at the exon-intron junction of exon 21 of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene responsible for ARHR2 (IVS21+1_3(GTA>CACC)). Subsequent analysis of mRNA revealed that this mutation caused skipping of exon 21 which created a premature stop codon in exon 22. These results indicate that genetic analysis is mandatory for the correct diagnosis of hereditary FGF23-related hypophosphatemic rickets. Because Enpp1 knockout mouse is a model of OPLL, this case also suggests that OPLL is associated with ARHR2.

FokI variant of vitamin D receptor gene and factors related to atherosclerosis associated with ossification of the posterior longitudinal ligament of the spine: a multi-hospital case-control study.

STUDY DESIGN: A sex- and age-matched case-control study with genotyping of the FokI variant of the vitamin D receptor gene (VDR) was carried out. OBJECTIVES: To facilitate the early prediction, prevention, and treatment of ossification of the posterior longitudinal ligament (OPLL) of the spine, we analyzed the FokI variant of VDR and past body mass indexes, histories of past illness, family history, and body pliability along with lifestyle factors. SUMMARY OF BACKGROUND DATA: Many possible genetic and environmental risk factors for OPLL have been suggested, including male sex, high body mass index, diabetes mellitus, trauma, hormonal imbalance, and dietary and sleeping habits and genetic variants. METHODS: Both a self-administered questionnaire and whole blood samples were obtained from 63 patients with OPLL and 126 sex-, age-, and hospital-matched controls free of backbone diseases were randomly selected from hospital patients. VDR genotyping was carried out using PCR-RFLP methods. After univariate analysis, multivariate and subgroup analyses according to the VDR genotype was applied to clarify the confounding relationship between VDR genotype and other possible risk factors. RESULTS: A multivariate analysis revealed that the VDR FF genotype, family history of myocardial infarction, high body mass index at age 40, long working hours, and working with night shift to be independent potent risk factors for OPLL. CONCLUSION: The risk of developing OPLL may possibly be reduced gradually and effectively by removing or minimizing the effect of such lifestyle factors one at a time through targeted preventive intervention.

Decrease in serum nucleotide pyrophosphatase activity in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is a prototype of a group of rheumatic diseases referred to as spondyloarthropathy. AS patients show marked ectopic ossification in the spine, occasionally resulting in so-called bamboo spine. Although a strong association with HLA-B27 has been reported, its aetiology remains undetermined. Another rheumatic disease, ossification of the posterior longitudinal ligament of the spine (OPLL), demonstrates ectopic ossification of the spinal ligaments very similar to that of AS. Recently, nucleotide pyrophosphatase (NPPS) was implicated in the aetiology of OPLL: an Npps mutation was found to cause OPLL in mice, and an association between a polymorphism of the human NPPS gene and OPLL was identified. The clinical similarities between AS and OPLL led us to hypothesize that NPPS may also be implicated in the aetiology of AS. To elucidate the role of NPPS in the pathogenesis of AS, we examined serum NPPS activity and the possible association of the NPPS gene with AS. METHODS: Forty-four Japanese patients with AS, 43 patients with OPLL, and age- and sex-matched normal volunteers took part in this study. We determined serum NPPS activity using high-performance liquid chromatography and examined the association between AS and NPPS using single nucleotide polymorphisms (SNPs) of the NPPS gene. RESULTS: Serum NPPS activity in AS patients was significantly decreased compared with the controls (P < 0.0001). However, there was no association between AS and NPPS gene SNPs. CONCLUSION: NPPS is implicated in the pathogenesis of AS.

Nucleotide pyrophosphatase gene polymorphism associated with ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a disease that causes paralysis by compressing the spinal cord. Based on the fact that the nucleotide pyrophosphatase (Npps) gene is responsible for ectopic ossification in ttw, an OPLL model mouse, the possibility was explored whether the human NPPS gene is associated with susceptibility to and severity of OPLL. First, we screened for single-nucleotide polymorphisms (SNPs) in the human NPPS locus using selected 25 OPLL patients with young onset (< 35 years old) or severe ossification (> 10 ossified vertebrae), and identified three novel SNPs in the locus. A case-control association study between 180 OPLL patients and 265 non-OPLL controls showed that one of these SNPs, IVS15-14T --> C substitution, was more frequently observed in OPLL patients (p = 0.022), especially in those with severe ossification (p < 0.0001) and young onset (p = 0.002), than in controls. A stratified study with the number of ossified vertebrae in OPLL patients revealed that IVS15-14T --> C substitution (p = 0.013) as well as young onset (p = 0.046) and female sex (p = 0.006) were associated with severe ossification. We conclude that the IVS15-14T --> C substitution in the human NPPS gene is associated not only with susceptibility to, but also with severity of OPLL.

Mutation in Npps in a mouse model of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common form of human myelopathy caused by a compression of the spinal cord by ectopic ossification of spinal ligaments. To elucidate the genetic basis for OPLL, we have been studying the ttw (tiptoe walking; previously designated twy) mouse, a naturally occurring mutant which exhibits ossification of the spinal ligaments very similar to human OPLL (refs 3,4). Using a positional candidate-gene approach, we determined the ttw phenotype is caused by a nonsense mutation (glycine 568 to stop) in the Npps gene which encodes nucleotide pyrophosphatase. This enzyme regulates soft-tissue calcification and bone mineralization by producing inorganic pyrophosphate, a major inhibitor of calcification. The accelerated bone formation characteristic of ttw mice is likely to result from dysfunction of NPPS caused by predicted truncation of the gene product, resulting in the loss of more than one-third of the native protein. Our results may lead to novel insights into the mechanism of ectopic ossification and the aetiology of human OPLL.

Involvement of insulin-like growth factor I in development of ossification of the posterior longitudinal ligament of the spine.

In order to investigate the pathogenesis of ossification of the posterior longitudinal ligament (OPLL) of the spine, we examined the distribution of insulin-like growth factor I (IGF-I) in the posterior longitudinal ligaments of OPLL patients, and analyzed the effects of IGF-I on the cultured spinal ligament cells. For that purpose we established eight varieties of OPLL and non-OPLL cell lines obtained from spinal ligaments of corresponding patients, respectively. In contrast to non-OPLL cases, all the OPLL cases were histologically shown to contain round-shaped cartilage-like cells in the transitional region from preossifying to ossifying ligaments, and these cells were strongly stained with an antibody for IGF-I. In the vicinity of preossifying cartilaginous tissues, ligament cells also had a rod-like appearance and were positive for IGF-I immunohistochemically. The effects of IGF-I on cultured spinal ligament cells were assayed by alkaline phosphatase (AP) activity, DNA synthesis, and the amounts of collagen produced. The number of OPLL cell lines that increased AP activity, responding to IGF-I irrespective of 1,25(OH)2D3, was significantly larger than that of non-OPLL cell lines, although IGF-I stimulated DNA and procollagen type I carboxyl-terminal peptide synthesis in most of both OPLL and non-OPLL cell lines. These data demonstrate the dominant expression of IGF-I in the posterior longitudinal ligaments of OPLL patients, and suggest that IGF-I preferentially induces osteogenic differentiation in OPLL cells rather than in non-OPLL cells. IGF-I, therefore, may be involved in the local ossification process of spinal ligaments observed in OPLL patients.

Effects of bone-seeking hormones on DNA synthesis, cyclic AMP level, and alkaline phosphatase activity in cultured cells from human posterior longitudinal ligament of the spine.

In a study of the osteogenesis capability of the human posterior longitudinal ligament of the spine, ligament cells were isolated and cultured. The effect of bone-seeking hormones, such as parathyroid hormone (PTH), calcitonin (CT), prostaglandin E2 (PGE2), and 1,25-dihydroxycholecalciferol [1,25-(OH)2D3], on the ligament cells was investigated with respect to DNA synthesis, adenosine-3',5'-cyclic monophosphate (cAMP) levels, alkaline phosphatase (ALP) activity, and acid phosphatase (ACP) activity. Cell lines obtained from nonossified sites in patients with ossification of the posterior longitudinal ligament of the spine (OPLL) were found to have several different phenotypic characteristics for osteoblasts: high ALP activity, PTH- and PGE2-stimulated increases in cAMP, and responses to both CT and 1,25-(OH)2D3. It is clear that proliferation and differentiation in such ligament cells are controlled by various types of bone-seeking hormones, and it was suggested that many cells with osteoblast-like characteristics are present. These results are considered important with respect to the etiology of OPLL, and an experimental system using cultured ligament cells appears to be useful in research on OPLL.