A functional RNAi screen for Runx2-regulated genes associated with ectopic bone formation in human spinal ligaments.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic ossification in the spinal ligaments, which enlarges with time and compresses the spinal cord, resulting in serious neurological symptoms. We previously reported that Runx2 expression was enhanced in spinal ligament cells from OPLL patients (OPLL cells). To clarify genes regulated by Runx2, Runx2 expression was first enhanced by culturing primary OPLL cells in osteogenic medium (OS induction) and then inhibited by siRNAs targeted to Runx2. DNA microarray demonstrated that in addition to chondrogenic factors such as connective tissue growth factor and cartilage oligomeric matrix protein, angiopoietin-1 was also significantly increased by OS induction and decreased by siRNAs for Runx2 in OPLL cells, suggesting that these genes are regulated by Runx2. However, these changes were not observed in non-OPLL control cells (from cervical spondylotic myelopathy patients). Furthermore, Runx2 was not decreased by siRNAs for angiopoietin-1. OS induction and RNAi inhibition of angiopoietin-1 expression was also observed in osteoblasts. Both siRNAs for Runx2 and angiopoietin-1 completely inhibited aggrecan-1 expression. These results suggest that angiopoietin-1 is downstream of Runx2 in both OPLL primary cells and osteoblasts. Angiopoietin-1 may play an important role in ectopic ossification.

Interaction between proteoglycans and alpha-elastin in construction of extracellular matrix of human yellow ligament.

One type of large proteoglycan and three types of small proteoglycans (decorin, decorin-subtype, and biglycan) were purified by chromatography, and alpha-elastin was isolated by alkali treatment from human yellow ligaments taken at the time of operation. The interaction of the proteoglycans with immobilized alpha-elastin on a sensor was analyzed by surface plasmon resonance, and we confirmed that each of the small proteoglycans exhibited a specific binding to alpha-elastin. The binding sites of small proteoglycans were contained in the protein cores. In addition, the differences in the interaction of the small proteoglycans with alpha-elastin of normal and ossified ligaments were compared. The interactions of the small proteoglycans with alpha-elastin of the ossified ligaments were lower than those with alpha-elastin of the normal ligaments. In the ossified ligaments, neodesmosine, one of the cross-linking amino acids, was significantly less than in the normal ligaments (p < .05).

An immunohistochemical evaluation of extracellular matrix components in the spinal posterior longitudinal ligament and intervertebral disc of the tiptoe walking mouse.

Ossification of the posterior longitudinal ligament (OPLL) in the spine is caused by systemic and/or regional factors affecting the regulation of osteocartilaginous formation and maintenance. The aims of this study were to elucidate the relationship between the degeneration of the intervertebral discs and changes in the posterior longitudinal ligament (PLL) in the tiptoe walking (ttw) mouse, an animal model of OPLL, and to analyze the sequential changes of the cells producing extracellular matrix components using immunohistochemical methods. At 6 weeks of age, the discs degenerated and the chondrocytes in the nucleus pulposus were positive for chondroitin-6-sulfate in the ttw mice. The fibroblasts in the PLL at the disc level were positively stained with type II and XI collagens. At 14 weeks, the discs herniated into the thickened PLL, and chondrocyte-like cells appeared in the PLL at vertebral endplate level. At 18 and 22 weeks, the number of chondrocyte-like cells increased in the PLL and expressed type I collagen. A potent regional factor causing OPLL in the ttw mice appears to be the initial degeneration and subsequent herniation of the nucleus pulposus. These sequential changes in the ttw mice were accelerated by administration of etidronate. It was suggested that etidronate stimulated the cartilaginous hyperplasia in the PLL of the ttw mice. It appeared as if the PLL transformed itself into cartilaginous tissue to repair the degeneration of the intervertebral disc.

Genomewide linkage and linkage disequilibrium analyses identify COL6A1, on chromosome 21, as the locus for ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament (OPLL) of the spine is a subset of "bone-forming" diseases, characterized by ectopic ossification in the spinal ligaments. OPLL is a common disorder among elderly populations in eastern Asia and is the leading cause of spinal myelopathy in Japan. We performed a genomewide linkage study with 142 affected sib pairs, to identify genetic loci related to OPLL. In multipoint linkage analysis using GENEHUNTER-PLUS, evidence of linkage to OPLL was detected on chromosomes 1p, 6p, 11q, 14q, 16q, and 21q. The best evidence of linkage was detected near D21S1903 on chromosome 21q22.3 (maximum Zlr=3.97); therefore, the linkage region was extensively investigated for linkage disequilibrium with single-nucleotide polymorphisms (SNPs) covering 20 Mb. One hundred fifty positional candidate genes lie in the region, and 600 gene-based SNPs were genotyped. There were positive allelic associations with seven genes (P<.01) in 280 patients and 210 controls, and four of the seven genes were clustered within a region of 750 kb, approximately 1.2 Mb telomeric to D21S1903. Extensive linkage disequilibrium and association studies of the four genes indicated that SNPs in the collagen 6A1 gene (COL6A1) were strongly associated with OPLL (P=.000003 for the SNP in intron 32 [-29]). Haplotype analysis with three SNPs in COL6A1 gave a single-point P value of.0000007. Identification of the locus of susceptibility to OPLL by genomewide linkage and linkage disequilibrium studies permits us to investigate the pathogenesis of the disease, which may lead to the development of novel therapeutic tools.

Role of prostaglandin I2 in the gene expression induced by mechanical stress in spinal ligament cells derived from patients with ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments, and mechanical stress has been suggested to play an important role in the progression of OPLL. To identify the genes that participate in OPLL, the differential display reverse transcription-polymerase chain reaction (RT-PCR) method was used. A 283-base pair cDNA fragment corresponding to prostaglandin I2 (PGI2) synthase was highly expressed in OPLL cells compared with non-OPLL cells. To examine the effect of mechanical stress on the expression of PGI2 synthase, cells were subjected to uniaxial cyclic stretch (0.5 Hz, 20% stretch), and PGI2 synthase mRNA expression was assessed by quantitative RT-PCR. Cyclic stretch induced an increase in PGI2 synthase in OPLL cells in a time-dependent manner, whereas no change was observed in non-OPLL cells. Cyclic stretch for 9 h also induced a 2.86x increase in PGI2 production. Beraprost (a stable PGI2 analog) and dibutyryl cAMP (a membrane-permeable cAMP analog) increased the mRNA expression of alkaline phosphatase (ALP) as a marker for osteogenic differentiation up to 240 and 200%, respectively, in OPLL cells, whereas no change was observed in non-OPLL cells. The increases in ALP mRNA induced by beraprost and cyclic stretch were both inhibited by SQ22536, a potent adenylate cyclase inhibitor. These data suggest that the increase in PGI2 synthase induced by mechanical stress plays a key role in the progression of OPLL, at least in part through the induction of osteogenic differentiation in spinal ligament cells via the PGI2/cAMP system.

Results of the latissimus dorsi and teres major tendon transfer on to the rotator cuff for brachial plexus palsy at birth.

Brachial plexus palsy at birth remains a serious problem. Although most cases resolve during the first few months by spontaneous regeneration, several operations have been used to correct the residual deformity. In the present study we describe the results of the latissimus dorsi and teres major tendons transfer on to the rotator cuff to improve shoulder function. Six patients were included in the study: three girls and three boys; four right shoulders, and two left. The types of palsy were four Erb's palsy (C5, C6) and two C5-C7 palsy. The median age at the time of operation was 11 years and 1 month and the median follow-up period was 54.2 months. Median preoperative passive external rotation was 51 degrees, and active abduction 67 degrees. Median postoperative active external rotation was 72 degrees, and postoperative active abduction 109 degrees. This procedure increased the ranges of external rotation and abduction, and provided considerable improvement in shoulder function.

Possible roles of CTGF/Hcs24 in the initiation and development of ossification of the posterior longitudinal ligament.

STUDY DESIGN: A biochemical and histochemical study investigating the role of CTGF/Hcs24 in the ossification of the posterior longitudinal ligament (OPLL) was conducted. OBJECTIVE: To clarify the involvement of CTGF/Hcs24 in ectopic bone formation in OPLL through endochondral ossification using human tissue. SUMMARY OF BACKGROUND DATA: Previous studies have shown that various cytokines are involved in the occurrence or development of ectopic bone formation in OPLL. Recently, the authors cloned an mRNA predominantly expressed in chondrocytes by differential display PCR and found that its gene, hcs24, is identical to that of connective tissue growth factor. It has been shown that CTGF/Hcs24 plays a major role in endochondral ossification. METHODS: Ossified ligament tissues were taken from seven male OPLL patients during surgery. Immunohistochemical staining was performed using an antibody specific for CTGF/Hcs24. Spinal ligament cells were isolated from five OPLL patients as well as five non-OPLL patients. The cells were incubated with recombinant human CTGF/Hcs24 or TGFbeta. The expression of ALP was analyzed by RT-PCR. For the effects of TGFbeta, the expression of CTGF/Hcs24 mRNA was analyzed. RESULTS: Immunohistochemical staining showed that chondrocytes in the transitional region from nonossified to ossified ligament were stained with an antibody against CTGF/Hcs24. It was found that CTGF/Hcs24 enhanced the expression ALP mRNA in OPLL cells, whereas the expression remained unchanged in non-OPLL cells. The expression of CTGF/Hcs24 mRNA in OPLL and non-OPLL cell lines was increased by TGFbeta, and there was no significant difference between the two groups. However, TGFbeta and CTGF/Hcs24 enhanced the expression of ALP mRNA only in OPLL cells. CONCLUSIONS: According to the study results, CTGF/Hcs24 may not only be an important factor in the development of endochondral ossification in OPLL, but may also be responsible for initiating osteogenesis in spinal ligament cells.

Long-standing nonunion of fractures of the lateral humeral condyle.

BACKGROUND: Patients with nonunion of a fracture of the lateral humeral condyle often have pain, instability, or progressive cubitus valgus deformity with tardy ulnar nerve palsy. However, some patients have minimal or no symptoms or disabilities. We evaluated patients with long-standing established nonunion of the lateral humeral condyle to correlate the clinical long-term outcome of this condition with the original fracture type. METHODS: Nineteen elbows in eighteen patients who were at least twenty years of age were evaluated. Fourteen patients were male, and four were female. The average age at presentation was 42.5 years. The average interval from the injury to the presentation of the symptoms of the nonunion was thirty-seven years. Patients were divided into two groups on the basis of the size of the fragment and the location of the fracture line. Group 1 included nine elbows with nonunion resulting from a Milch Type-I injury, and Group 2 included ten elbows with a nonunion resulting from a Milch Type-II injury. Evaluations were performed with use of radiographic examination, clinical assessment, and calculation of the Broberg and Morrey score. RESULTS: Symptoms were seen more frequently in Group 1 than in Group 2. The range of flexion in Group 1 (range, 60 degrees to 145 degrees; average, 99 degrees) was more restricted than that in Group 2 (range, 100 degrees to 150 degrees; average, 129 degrees) (p = 0.0078). The functional score in Group 2 was significantly higher than that in Group 1 (p = 0.03). CONCLUSION: Disabling symptoms only rarely developed in Group-2 patients. Occasionally, however, these patients do present with clinically detectable dysfunction of the ulnar nerve. In contrast, pain, instability, and loss of range of motion as well as ulnar nerve dysfunction developed in Group 1. For this reason we think that a nonunion of a Milch Type-I fracture should be treated as soon as possible after injury, preferably before the patient reaches skeletal maturity.

Adult scoliosis in syringomyelia associated with Chiari I malformation.

STUDY DESIGN: In adult syringomyelia associated with Chiari I malformation, the spinal deformity, the configuration of cerebellar tonsillar descent, the configuration of syrinx, and the clinical evaluation before and after surgery were investigated. OBJECTIVES: To investigate the characteristics of the scoliosis in syringomyelia associated with Chiari I malformation. SUMMARY OF BACKGROUND DATA: In previous studies, the clinical characteristics of pediatric scoliosis associated with syringomyelia have been reported. METHODS: In this study, 42 patients with syringomyelia were treated. All the patients were 20 years of age or older. They were divided into three groups: Group 1 comprising those without scoliosis, Group 2 composed of those with scoliosis of 10 degrees or more but less than 20 degrees, and Group 3 consisting of those with scoliosis of 20 degrees or more. Investigations conducted with the three groups included determining the curve patterns of scoliosis, the degree of thoracic kyphosis, the configuration of cerebellar tonsillar descent, the configuration of syrinx, the morbidity period, and the clinical evaluation before and after surgery. RESULTS: There were 12 patients in Group 1, 21 patients in Group 2, and 9 patients in Group 3. The concomitant rate of adult syringomyelia with scoliosis was 71.4%. As scoliosis advanced, the kyphotic angle also increased. The concordance in laterality between the cerebellar tonsil and curve convex was 70%. Findings showed that the more advanced the scoliosis was, the more aggravated the neurologic symptoms were, and the poorer the surgical outcomes tended to be. CONCLUSIONS: In adult syringomyelia with scoliosis, the morbidity period is long, the syrinx is long, the neurologic symptoms are aggravated, and the surgical outcomes tend to be poor.

Large-scale screening for candidate genes of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is the predominant myelopathy among Japanese, and is usually diagnosed by ectopic bone formation in the paravertebral ligament in Japanese and other Asians. To detect genetic determinants associated with OPLL, we performed an extensive nonparametric linkage study with 126 affected sib-pairs using markers for various candidate genes by distinct analyses, SIBPAL and GENEHUNTER. Eighty-eight candidate genes were selected by comparing the genes identified by complementary DNA (cDNA) microarray analysis of systematic gene expression profiles during osteoblastic differentiation of human mesenchymal stem cells with the genes known to be involved in bone metabolism. Of the 24 genes regulated during osteoblastic differentiation, only one, the alpha B crystalline gene, showed evidence of linkage (p = 0.016, nonparametric linkage [NPL] score = 1.83). Of 64 genes known to be associated with bone metabolism, 7 showed weak evidence of linkage by SIBPAL analysis (p < 0.05): cadherin 13 (CDH13), bone morphogenetic protein 4 (BMP4), proteoglycan 1 (PRG1), transforming growth factor beta 3 (TGFb3), osteopontin (OPN), parathyroid hormone receptor 1 (PTHR1), and insulin-like growth factor 1 (IGF1). Among these genes, BMP4 (NPL = 2.23), CDH13 (NPL = 2.00), TGFb3 (NPL = 1.30), OPN (NPL = 1.15), and PTHR1 (NPL = 1.00) showed evidence of linkage by GENEHUNTER. Only BMP4 reached criteria of suggestive evidence of linkage. Because this gene is a well-known factor in osteogenetic function, BMP4 should be screened in further study for the polymorphism responsible.

Akaike's information criterion for a measure of linkage disequilibrium.

The extent and distribution of linkage disequilibrium ( LD) in humans is a current topic especially for gene mapping of complex diseases. Akaike's information criterion ( AIC) was applied to estimate LD and compared with other standard LD measures, D' and r(2). By comparison of an independent model (IM; linkage equilibrium) and a dependent model (DM; linkage disequilibrium), the parsimonious model is the one with the smaller AIC score. Therefore, the extent of LD by AIC is expressed as AIC( IM) -- AIC( DM)( AIC( LD)). A total of 39 single-nucleotide polymorphisms on a 1.6-Mb region of chromosome 21 q22 were identified, and genotyped in 192 Japanese individuals. All possible pairs were analyzed to estimate LD and the analyses were compared. AIC( LD) became highly positive as the D' value increased and was negative at D' values of around 0.2. Because a negative value of AIC( LD) implies linkage equilibrium, D' values below 0.2 should be regarded as linkage equilibrium. The LD estimate by AIC yielded results similar to those obtained by r(2), indicating that AIC( LD) would be useful for fine gene mapping.