Seiko Harata

Genetic mapping of ossification of the posterior longitudinal ligament of the spine.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is recognized as a common disorder among Japanese and throughout Asia. Estimates of its prevalence are in the range of 1. 9%-4.3%. Although its etiology is thought to involve a multiplicity of factors, epidemiological and family studies strongly implicate genetic susceptibility in the pathogenesis of OPLL. In this study we report an identification of a predisposing locus for OPLL, on chromosome 6p, close to the HLA complex. The evidence for this localization is provided by a genetic-linkage study of 91 affected sib pairs from 53 Japanese families. In this sib-pair study, D6S276, a marker lying close to the HLA complex, gives evidence for strongly significant linkage (P = .000006) to the OPLL locus. A candidate gene in the region, that for collagen 11A2, was analyzed for the presence of molecular variants in affected probands. Of 19 distinct variants identified, 4 showed strong statistical associations with OPLL (highest P = .0004). These observations of linkage and association, taken together, show that a genetic locus for OPLL lies close to the HLA region, on chromosome 6p.

Spinous process-splitting laminoplasty using hydroxyapatite spinous process spacer.

Between February 1986 and August 1989, 45 patients with cervical myelopathy were treated by spinous process-splitting laminoplasty. Hydroxyapatite intraspinous spacers were used to maintain the enlargement of the cervical spinal canal. The shape of this spacer is trapezoidal. After sagittal splitting of the spinous process, the spacer was inserted between the two haives and affixed with the wire. Histologic study showed there was good fusion between the spacer and bone. In all cases, good enlargement of the cervical spinal canal was achieved. Spacer displacement, wire breakage, and postoperative infection were not seen. There was no postoperative neurologic deterioration. Computed tomography showed that the width of the cervical spinal canal was maintained.

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.

Uniaxial cyclic stretch induces osteogenic differentiation and synthesis of bone morphogenetic proteins of spinal ligament cells derived from patients with ossification of the posterior longitudinal ligaments

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To elucidate this mechanism, we investigated the effects of in vitro sinusoidal cyclic stretch (120% peak to peak, at 1 Hz) on cultured spinal ligament cells derived from OPLL and non-OPLL patients. The mRNA expressions of alkaline phosphatase (ALP), osteopontin, bone morphogenetic protein (BMP)-2, BMP-4, and BMP receptors as well as ALP activity in cell layers and production of BMPs into the conditioned medium were significantly increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. A stretch-activated Ca(2+) channel blocker, Gd(3+), the voltage-dependent L-type Ca(2+) channel blockers diltiazem and nifedipine, and Ca(2+)-free medium suppressed stretch-induced ALP activity, which suggests a role of Ca(2+) influx in the signal transduction of mechanical stress to the osteogenic response of OPLL cells. Our study provides first evidences that mechanical stress plays a key role in the progression of OPLL through the induction of osteogenic differentiation in spinal ligament cells and the promotion of the autocrine/paracrine mechanism of BMPs in this lesion.

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° or more but less than 20°, and Group 3 consisting of those with scoliosis of 20° 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.

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 TGF&bgr;. The expression of ALP was analyzed by RT-PCR. For the effects of TGF&bgr;, 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 TGF&bgr;, and there was no significant difference between the two groups. However, TGF&bgr; 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.

Human retinoic X receptor β : Complete genomic sequence and mutation search for ossification of posterior longitudinal ligament of the spine

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the ligament. OPLL is a very common disorder, in fact it constitutes the leading cause of myelopathy among Japanese. In the previous report, we provided the genetic linkage evidence that the genetic susceptibility of OPLL mapped to HLA complex of chromosome 6. As a candidate gene approach, retinoic X receptor β (RXRβ), assigned to chromosome 6p21.3 adjacent to HLA class II, was analyzed for a possible causality. To start screening for the molecular variants of RXRβ in OPLL subjects, we first obtained P1 phage genomic clones containing the entire human RXRβ and elucidated the genomic organization of the gene. The human RXRβ is composed of 10 exons spanning over 6.2 kb of genomic DNA. Sequence analysis of the promoter region revealed a GC‐rich sequence without TATA motif. We have identified three distinct molecular variants, one was in exon 10 and two were in the intergenic region between RXRβ and collagen 11A2 (COL11A2). Two variants in the intergenic region, 3′ end + 140 and 3′ end + 561, exhibit statistically significant associations with OPLL in case‐control study (p = 0.0028 for 3′ end + 140 and p = 0.034 for 3′ end + 561). These results indicate that the genetic causality of OPLL lies within or close to the RXRβ/COL11A2 locus.

Uni-axial Cyclic Stretch Induces Cbfa1 Expression 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. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin β1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin β1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.

Gender-specific haplotype association of collagen α2 (XI) gene in ossification of the posterior longitudinal ligament of the spine

AbstractAmong Japanese, ossification of the posterior longitudinal ligament of the spine (OPLL) is a leading cause of myelopathy, showing ectopic bone formation in the paravertebral ligament. We have provided genetic evidence that the collagen α2 (XI) (COL11A2) locus of chromosome 6 constitutes susceptibility for OPLL. Five distinct single nucleotide polymorphisms (SNPs), identified in COL11A2, were combined to construct possible haplotypes by the use of a maximum likelihood program. Estimated haplotype frequency was compared in OPLL patients and non-OPLL controls. We report a gender-specific association of the COL11A2 haplotype with OPLL. The frequency of the most commonly observed haplotype was significantly higher in male patients (P = 0.0003) compared with controls, but not in female patients (P = 0.21). OPLL is predominantly observed in males, with a prevalence ratio of 2 : 1, and our gender-specific associations indicate that genetic factors involving COL11A2 play a specific role in the etiology of OPLL exclusively in males.

Anterior cervical fusion using porous hydroxyapatite ceramics for cervical disc herniation. a two-year follow-up.

BACKGROUND CONTEXT The Smith-Robinson Method (SR), which employs autogenous bone, is the current standard for anterior cervical fusion (AF) surgery. However, autogenous bone has graft-related complications and morbidity, and harvesting it increases trauma and risk to the patient. The use of hydroxyapatite ceramic (HAP) inserts may provide a superior alternative. PURPOSE To determine the efficacy of using HAP in AF. STUDY DESIGN/SETTING A retrospective study of patients who had AF surgery with wide decompression and porous HAP inserts used to treat cervical disc herniation (CHD). PATIENT SAMPLE We evaluated 36 patients who had single-level AF using HAP for CHD, without internal fixations, clinically and radiographically with a minimum follow-up of 2 years. There were 25 men and 11 women, with an average age of 49 years (age range, 24-78 years). Preoperative diagnosis included 25 cases with myelopathy and 11 cases with radiculopathy. OUTCOME MEASURES We established four grades to classify the degree of bony fusion between the HAP and vertebra, based on any motion at the fused segment, any radiolucent zones (RZ) between vertebral bodies and the grafted HAP, and anterior or posterior bone formations on grafted HAPs. We evaluated the severity of myelopathy by applying the Japan Orthopaedic Association (JOA) scoring system. We evaluated the surgical outcome of the myelopathy patients using the Hirabayashi recovery rating, and for the radiculopathy patients, we used the Herkowitz criteria. METHODS We retrospectively reviewed the radiographic and clinical records of all 36 patients from surgery up to periods ranging from 2 to 7 years after surgery, with the average period of follow-up being 4.5 years. We systematically classified the degree of bony fusion into four grades ranging from Grade 1 nonunion to Grade 4 complete union. RESULTS None of the subjects showed Grades 1 and 2 fusion. Eleven percent of the cases showed Grade 3 and 89% showed Grade 4. Loss of height of the fused segment was observed in 29 cases with an average of 1.6 mm. A decrease of lordotic angle of the fused segment was observed in six cases with an average of 2.3 degrees. Four cases revealed cracked HAP inserts but achieved Grade 4 bone fusion. There was no evidence of collapse or displacement of HAPs. The results of the 11 radiculopathy patients were excellent in 10 cases and good in the remaining case. The recovery rate of the 25 myelopathy patients was 73.0%. CONCLUSIONS Our method of anterior cervical fusion surgery using porous HAP inserted into resected end plates, combined with a wide decompression procedure, had clinical and radiographic results so satisfactory that we conclude that it can effectively replace the use of autogenous bone for treating cervical disc herniation.