Teppei Suzuki

A genome-wide association study identifies common variants near LBX1 associated with adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis is a pediatric spinal deformity affecting 2–3% of school-age children worldwide. Genetic factors have been implicated in its etiology. Through a genome-wide association study (GWAS) and replication study involving a total of 1,376 Japanese females with adolescent idiopathic scoliosis and 11,297 female controls, we identified a locus at chromosome 10q24.31 associated with adolescent idiopathic scoliosis susceptibility. The most significant SNP (rs11190870; combined P = 1.24 × 10−19; odds ratio (OR) = 1.56) is located near LBX1 (encoding ladybird homeobox 1). The identification of this susceptibility locus provides new insights into the pathogenesis of adolescent idiopathic scoliosis.

Genetic variants in GPR126 are associated with adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) is the most common pediatric skeletal disease. We previously reported a locus on chromosome 10q24.31 associated with AIS susceptibility in Japanese using a genome-wide association study (GWAS) consisting of 1,033 cases and 1,473 controls. To identify additional AIS-associated loci, we expanded the study by adding X-chromosome SNPs in the GWAS and increasing the size of the replication cohorts. Through a stepwise association study including 1,819 cases and 25,939 controls, we identified a new susceptibility locus on chromosome 6q24.1 in Japanese (P = 2.25 × 10−10; odds ratio (OR) = 1.28). The most significantly associated SNP, rs6570507, was in GPR126 (encoding G protein–coupled receptor 126). Its association was replicated in Han Chinese and European-ancestry populations (combined P = 1.27 × 10−14; OR = 1.27). GPR126 was highly expressed in cartilage, and the knockdown of gpr126 in zebrafish caused delayed ossification of the developing spine. Our results should provide insights into the etiology and pathogenesis of AIS.

Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration

IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

Fas ligand expression on human nucleus pulposus cells decreases with disc degeneration processes

BackgroundThe intervertebral disc has been reported to be an immunologically privileged environment, possibly mediated by Fas ligand (FasL) expression. On the other hand, recent studies have shown the infiltration of host immune cells into the degenerated disc, which may indicate the failure of the immune-privilege feature of the disc with degeneration. However, the relationship between FasL expression and disc degeneration is still unclear. Therefore, the purpose of this study was to clarify the relationship between FasL expression and disc degeneration.MethodsTen human degenerated disc specimens were obtained from spondylolisthesis patients and ten nondegenerated discs from idiopathic scoliosis patients during surgical procedures. Immunohistochemical staining was performed to determine the presence of FasL in cross-sections of those discs. Parts of the disc tissues were used to examine FasL expression quantitatively with Western blot analysis. To examine whether the change in FasL expression was influenced by aging, an animal study comparing the discs from young and old rats were performed using magnetic resonance imaging (MRI) and real-time polymerase chain reaction (PCR) assessment.ResultsNucleus pulposus cells showed strong positive staining for FasL in all specimens examined. Quantitative examination demonstrated a significant decrease in FasL expression in the degenerated group compared with the nondegenerated group (average 67.6%, P<0.05). MRI showed no significant differences in the grade of disc degeneration between young and old rats, and also no significant difference in FasL mRNA in real-time PCR assay.ConclusionsThe current results indicate that FasL and its potential mechanism of immunological privilege could influence the protection of the intervertebral disc against degeneration.

Lack of association between adolescent idiopathic scoliosis and previously reported single nucleotide polymorphisms in MATN1, MTNR1B, TPH1, and IGF1 in a Japanese population.

Adolescent idiopathic scoliosis (AIS) is a spinal deformity most commonly arising in apparently healthy girls around puberty. AIS has a strong genetic predisposition. Several genetic associations between AIS and single nucleotide polymorphisms (SNPs) have been reported; common SNPs in the genes for matrilin 1 (MATN1), melatonin receptor 1B (MTNR1B), tryptophan hydroxylase 1 (TPH1), and insulin‐like growth factor 1 (IGF1) are reported to be associated with AIS in Chinese. However, these associations have not been replicated so far. To confirm the associations, we compared these SNPs with AIS predisposition and curve severity in a population of Japanese females consisting of 798 AIS patients and 1,239 controls. All the subjects were genotyped using the PCR‐based Invader assay. We found no association of any of the SNPs with AIS predisposition or curve severity. Considering the statistical power and sample size of the present study, we concluded that these SNPs are not associated with either AIS predisposition or curve severity in Japanese.

Replication study of the association between adolescent idiopathic scoliosis and two estrogen receptor genes.

Adolescent idiopathic scoliosis (AIS) is a common disorder with a strong genetic predisposition. Associations between AIS and common single nucleotide polymorphisms (SNPs) in estrogen receptor genes have been reported. rs9340799 in the gene for estrogen receptor α (ESR1) is reported to be associated with curve severity in Japanese and with AIS predisposition and curve severity in Chinese. In addition, rs1256120 in the gene for estrogen receptor β (ESR2) is reported to be associated with AIS predisposition and curve severity in Chinese. However, the sample sizes of these previous studies were small, and the associations of these SNPs have not been replicated. To examine the association between AIS and estrogen receptor genes, we investigated the association of rs9340799 and rs1256120 with AIS predisposition and curve severity using a large Japanese population, consisting of 798 AIS patients and 637 sex‐matched controls. We found no association of either SNP with AIS predisposition or curve severity in the Japanese population. Considering the statistical power of the present study and the limitations of the previous reports, we conclude that the associations of rs9340799 and rs1256120 with AIS predisposition and curve severity are negative.

Gene therapy approach for disc degeneration and associated spinal disorders

Disc degeneration is deeply associated with many spinal disorders and thus has a significant clinical impact on society. The currently available surgical treatment often necessitates removing a pathological disc and spinal fusion. However, it is also well known that these surgical treatments have many potential problems including invasion and cost. Therefore, biological approaches for regenerating these pathological discs have received much attention. Gene therapy is one of these biological approaches. Gene therapy involves the transfer of genes to cells so the recipient cells express these genes and thereby synthesize the RNA and protein they encode in a continuous fashion. One of the significant advantages of gene therapy is that we can expect a lasting duration of biological effect which is potentially beneficial for most disc degeneration associated disorders, as they are, by nature, chronic conditions. Originally, gene therapy was mediated by viral vectors, but recent technological progress has enabled us to opt for non-virus-mediated gene therapy for the disc. Furthermore, the development of the RNA interference technique has enabled us to down-regulate a specific gene expression in the disc opening the door for a new generation of intradiscal gene therapy.

Risk factors for complications associated with growing-rod surgery for early-onset scoliosis

Study Design. A retrospective multicenter study. Objective. To identify risk factors for postoperative complications associated with growing-rod (GR) surgery for early-onset scoliosis (EOS). Summary of Background Data. Results and complications of GR surgery for EOS have not been adequately studied. Methods. We evaluated clinical and radiographical results from 88 patients with EOS who underwent GR surgery in 12 spine centers in Japan. The mean age at the time of initial surgery was 6.5 ± 2.2 years (range, 1.5–9.8 yr) and the mean follow-up period was 3.9 ± 2.6 years (range, 2.0–12.0 yr). Risk factors for postoperative complications were analyzed using binomial multiple logistic regression analysis. We considered the potential factors of sex, age, number of rod-lengthening procedures, whether a pedicle screw foundation was used, the uppermost level of the proximal foundation and lowermost level of the distal foundation, Cobb angles of the proximal thoracic, main thoracic, and lumbar curves, and the kyphosis angles in the proximal, main thoracic, thoracolumbar, and lumbar spine. Kaplan-Meier analysis was used to determine the complication-free survival rate of GR surgery as a function of the number of surgical procedures. Results. Complications affected 50 of the patients (57%) and were associated with 119 of 538 surgical procedures, with 86 implant-related failures (72%), 19 infections (16%), 3 neurological impairments (3%), and 11 other complications. The most frequent implant-related failure was dislodged implant (71%) and 95% of the dislodgements occurred at the proximal foundation. Kaplan-Meier analysis demonstrated a linear decrease in complication-free rates as the number of rod-lengthening procedures increased. Binomial multiple logistic regression analysis found the following significant independent risk factors: 6 or more rod-lengthening procedures (odds ratio [OR], 6.534), an increase of every 20° in the proximal thoracic Cobb angle (OR, 3.091), and an increase of every 25° in the lumbar lordosis angle (OR, 2.607) in the preoperative condition. Conclusion. Increases in the upper thoracic scoliotic curve, thoracic kyphosis, and number of rod-lengthening procedures are positively associated with an increased risk of complications after GR surgery for EOS. Level of Evidence: 4

A Functional SNP in BNC2 Is Associated with Adolescent Idiopathic Scoliosis

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10(-13); odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS.

Postoperative distal adding-on and related factors in Lenke type 1A curve.

Study Design. A retrospective, multicenter study. Objective. To investigate the occurrence of and factors related to postoperative adding-on in Lenke type 1A curve. Summary of Background Data. Although several studies have investigated factors associated with adding-on in Lenke type 1A curve, these factors have not been elucidated in a large study population. Methods. This study included 112 patients who were followed more than 2 years after undergoing selective posterior thoracic fusion surgery for Lenke Type 1A curve (8 males, 104 females; mean age at surgery, 16.1 yr; mean follow-up, 3.6 yr). The lower instrumented vertebra (LIV) was T12 in 22 patients, L1 in 55, L2 in 32, and L3 in 3. Distal to the main thoracic curve, the end vertebra, neutral vertebra, stable vertebra (SV), and the last vertebra touching the central sacral vertical line (last touching vertebra, LTV) were determined. The occurrence and factors associated with distal adding-on were investigated. Results. The mean Cobb angle and apical translation of the main thoracic curve were 54.6° ± 9.6° and 53.1 ± 20.4 mm before surgery, and 14.2 ± 7.4 and 16.2 ± 12.7 at follow-up, respectively. Distal adding-on was observed in 21 patients (18.8%) at follow-up. Univariate analyses identified several factors significantly associated with adding-on, including the preoperative proximal thoracic curve, the apical translation of the main thoracic curve, Miyanjis subclassification, the postsurgical proximal and main thoracic curves, the postsurgical apical translation of the main thoracic curve, the correction rate of the main thoracic curve and the clavicle angle immediately after surgery and at follow-up, and the difference in levels between the LIV and the end vertebra, neutral vertebra, LTV, and stable vertebra. Logistic regression analysis showed that the apical translation of the main thoracic curve immediately after surgery (apical translation >25 mm, odds ratio: 10.7, 95% confidence interval: 3.1–37.0, P = 0.001) and the difference in levels between LIV and LTV (LIV-LTV) (LIV-LTV <0, odds ratio: 6.7, 95% confidence interval: 1.9–23.9, P = 0.003) were significantly associated with adding-on. Conclusion. Since the residual apical translation of the main thoracic curve and the lowest instrumented vertebra more cranial to the last touching vertebra were significantly associated with adding-on, surgeons may need to obtain the maximum reduction of the apical translation of the main thoracic curve and to extend the LIV at least to the LTV to avoid postoperative adding-on. Level of Evidence: N/A