Decrease in serum nucleotide pyrophosphatase activity in ankylosing spondylitis
K. Mori1,3,,
T. Chano2,5,
T. Ikeda4,
S. Ikegawa4,
Y. Matsusue1,
H. Okabe2 and
Y. Saeki3
1 Department of Orthopaedic Surgery,
2 Department of Clinical Laboratory Medicine and
3 Department of Basic Science for Health and Nursing, Shiga University of Medical Science (SUMS), Tsukinowa-cho, Seta, Otsu, Shiga 520-2192,
4 Laboratory for Bone and Joint Diseases, SNP Research Center, RIKEN (The Institute of Physical and Chemical Research), 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 and
5 PRESTO, Japan Science and Technology Corporation, Japan
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Abstract
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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.
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Introduction
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Ankylosing spondylitis (AS) is a prototype of a group of rheumatic diseases referred to as spondyloarthropathy [1]. AS is characterized by the involvement of the sacroiliac joints and the vertebral column, and general enthesopathy is also peculiar to AS. AS shows marked ectopic ossification in the spine, occasionally resulting in so-called bamboo spine during its final stage. Although a strong association of HLA-B27 with AS has been demonstrated [2], its aetiology remains largely unknown.
Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common form of human myelopathy that results from ectopic bone formation in the ligament of the spine [3]. Recently, nucleotide pyrophosphatase (NPPS: EC 3.6.1.9.) has been implicated in the aetiology of OPLL. NPPS activity, as exemplified by liver/bone/kidney alkaline phosphatase, could modulate the mineralization of skeletal tissues by generating inorganic pyrophosphate (PPi) [4]. In tiptoe walking (ttw) mice, an excellent model of human OPLL, a nonsense mutation of the mouse NPPS gene (Npps) was found to be responsible for the phenotype [5]. Furthermore, association of a sequence polymorphism of the NPPS gene with OPLL was identified in a casecontrol study [6].
OPLL has common features with AS in terms of ectopic ossification, such as ossification of spinal ligaments and entheses. The clinical similarities between AS and OPLL led us to hypothesize that NPPS may also be implicated in the aetiology of AS. Thus, to elucidate the role of NPPS in the pathogenesis of AS, we examined serum NPPS activity and the association of the NPPS gene with AS.
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Materials and methods
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Subjects
After they had given informed consent, 44 Japanese male AS patients, who fulfilled the modified New York criteria for AS [7], and 43 OPLL patients (32 males, 11 females) were recruited in this study. As controls, age- and sex-matched normal volunteers without liver dysfunction also took part. The study protocol was approved by the ethics committee of each participating institution. Serum was collected from peripheral blood and analysed for its NPPS activity. Also, genomic DNA was extracted from peripheral blood, using a standard method and subjected to analysis.
Assay of NPPS activity in serum
Analysis of serum NPPS was performed according to a method reported previously [8, 9], with slight modifications. Briefly, NPPS activity was determined using uridine diphosphate (UDP)-glucose as substrate. Ten microlitres of 1 M Tris buffer (pH 9.0), 1 µl of UDP-glucose, and 79 µl of distilled water were preincubated at 37°C. Then 10 µl of each serum sample was added and the reaction mixture was incubated at 37°C for 30 min. As a control, distilled water was used instead of serum. To stop the reaction, 5 µl of 2 N HCl was added to each sample, and the samples were kept at 4°C. Immediately, each 10 µl sample was subjected to high-performance liquid chromatography (HPLC) assay. The HPLC system used in this study included a high-pressure piston pump (LC-6A; Shimadzu, Kyoto, Japan), a reverse-phase column (5C18, 4.6x150 mm; Cosmosil, Nakarai, Japan) with a guard column (10C18, 4.6x50 mm; Cosmosil), and a UV-VIS spectrophotometric detector (SPD-6AV, Shimadzu). A high-pressure piston pump supplied the mobile phase at a rate of 1 ml/min using 0.1 M Na2HPO4 buffer. The mobile phase for elution was completed in 10 min. This system permitted the separation of uridine monophosphate (UMP) and UDP-glucose and could measure the peak height of the 260 nm absorption, which corresponded to UMP detected by the UV-VIS spectrophotometer. One unit of NPPS activity was defined as the amount of enzyme needed to catalyse the formation of UMP at an initial velocity of 1.00 µmol/min. Specific activity was expressed as U/mg of protein. Protein was determined by the biuret method.
Association study
Two common sequence polymorphisms in the NPPS gene [6] were tested for association with AS. One is in the promoter region (596T
C) and the other is in exon 4 (c.533A
C) of the NPPS gene. The 596T
C region was examined with a TaqMan method (ABI) and the c.533A
C region was subjected to PCR-RFLP (polymerase chain reactionrestriction fragment length polymorphism) analysis according to a method reported previously [6, 10].
Statistical analysis
StatView-J 4.02 (Abacus Concepts, Berkeley, CA, USA) was used for analyses. The MannWhitney U-test and the
2 test were applied when appropriate. P-values less than 0.05 were regarded as significant.
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Results
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Serum assay
To our knowledge, there is no report describing serum NPPS activity in a sufficient number of normal subjects. We measured serum NPPS activity in 156 normal subjects (77 males, 79 females). There was a significant difference in serum NPPS activity between the sexes (mean±S.D., 132±41x10-6 U/mg protein in males and 148±39x10-6 U/mg protein in females, P=0.01) (Fig. 1
). The serum NPPS activity in AS patients was significantly lower than in age- and sex-matched controls (Table 1
). In contrast, there was no significant difference in NPPS activity between OPLL patients and the control group. No significant difference in NPPS activity was found among subgroups of AS patients differing in clinical and biochemical parameters (bamboo spine, C-reactive protein level and HLA-B27; data not shown).
Association study
Forty-three DNA samples from AS patients were subjected to analysis, and the allele and genotype frequencies of the two common SNPs of the NPPS gene were compared between AS patients and the controls, as described previously [6]. We could not find any significant differences in the frequencies (Table 2
).
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Discussion
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We found that serum NPPS activity was decreased in normal male subjects. We also found that serum NPPS activity was decreased in AS patients but not in OPLL patients. The finding suggests a potential role of NPPS in the pathogenesis of AS. Clinical differences have been noted between the sexes. For example, male AS has been found to involve mainly the axial skeleton, in contrast to female AS, which frequently presents with peripheral arthritis [11], and male preponderance has also been noted in AS [12]. The decreased level of serum NPPS activity in males may be related to these clinical differences between the sexes in AS, though the cause of the decrease remains to be determined. We investigated the association of the NPPS gene with AS but obtained a negative result. Although our association study was limited in terms of the numbers of samples and markers, it is unlikely that a change in the NPPS gene itself is a cause of AS.
The role of NPPS in the pathogenesis of ectopic ossification remains unknown. Decreased NPPS activity could result in ectopic ossification: NPPS elaborates PPi via its phospholipase I/nucleotide pyrophosphatase activity [13]. PPi is a major inhibitor of calcification and mineralization [14], and therefore a decrease can cause ectopic ossification. Lower levels of NPPS activity and PPi have been found in ttw mice [15]. Moreover, a decrease in PPi was found in ank (progressive ankylosis) mice, another excellent model of ectopic ossification [16].
Alternatively, NPPS could inhibit ectopic ossification through the regulation of growth factors, such as transforming growth factor-ß and fibroblast growth factor, which have been implicated in bone formation [14, 17, 18]. Furthermore, through its role as an inhibitor of insulin-receptor tyrosine kinase [19], NPPS has been shown to inhibit the action of insulin, a stimulator of bone formation [20]. Additional studies are needed to elucidate the primary cause of the decrease in NPPS as well as its role in the pathogenesis in AS.
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Acknowledgments
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We thank Drs H. Inoue, K. Shichikawa, S. Hukuda, K. Inoue and T. Ushiyama and the members of the Japanese Association of Ankylosing Spondylitis Patients (AS Tomonokai) for their assistance in collecting blood samples.
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Notes
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Correspondence to: K. Mori, Shiga University of Medical Science, Tsukinowa-cho, Seta, Otsu, Shiga 520-2192, Japan. E-mail: kanchi{at}belle.shiga_med.ac.jp 
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Submitted 25 May 2001;
Accepted 28 May 2002