Are Copy Number Variants Associated With Adolescent Idiopathic Scoliosis?

Background

Adolescent idiopathic scoliosis (AIS) is a complex genetic disorder that causes spinal deformity in approximately 3% of the population. Candidate gene, linkage, and genome-wide association studies have sought to identify genetic variation that predisposes individuals to AIS, but the genetic basis remains unclear. Copy number variants are associated with several isolated skeletal phenotypes, but their role in AIS, to our knowledge, has not been assessed.

Questions/Purposes

We determined the frequency of recurrent copy number rearrangements, chromosome aneuploidy, and rare copy number variants in patients with AIS.

Methods

Between January 2010 and August 2014, we evaluated 150 patients with isolated AIS and spinal curvatures measuring 10° or greater, and 148 agreed to participate. Genomic copy number analysis was performed on patients and 1079 control subjects using the Affymetrix^® Genome-wide Human SNP Array 6.0. After removing poor quality samples, 143 (97%) patients with AIS were evaluated for copy number variation.

Results

We identified a duplication of chromosome 1q21.1 in 2.1% (N = 3/143) of patients with AIS, which was enriched compared with 0.09% (N = 1/1079) of control subjects (p = 0.0057) and 0.07% (N = 6/8329) of a large published control cohort (p = 0.0004). Other notable findings include trisomy X, which was identified in 1.8% (N = 2/114) of female patients with AIS, and rearrangements of chromosome 15q11.2 and 16p11.2 that previously have been associated with spinal phenotypes. Finally, we report rare copy number variants that will be useful in future studies investigating candidate genes for AIS.

Conclusions

Copy number variation and chromosomal aneuploidy may contribute to the pathogenesis of adolescent idiopathic scoliosis.

Clinical Relevance

Chromosomal microarray may reveal clinically useful abnormalities in some patients with AIS.     

HOXB9 Expression Promoting Tumor Cell Proliferation and Angiogenesis Is Associated with Clinical Outcomes in Breast Cancer Patients

Background

Studies have suggested that HOXB9 expression in breast cancer cells promotes cellular invasiveness, metastatic ability, and tumor neovascularization in the surrounding tissue in in vitro and in vivo assays. These findings imply that HOXB9 overexpression may alter tumor-specific cell fates and the tumor stromal microenvironment, contributing to breast cancer progression. The objective of this study was to analyze whether these results could be applied to clinical practice.

Methods

A total of 141 consecutive, invasive ductal carcinoma patients who underwent surgical treatment were examined. Immunohistochemical staining was performed to evaluate the expression of HOXB9, Ki-67, CD31, and CD34, and the association of tumor proliferation and angiogenesis with HOXB9 expression was analyzed.

Results

Of the 141 tumor specimens immunostained for HOXB9, 69 (48.9%) stained positive. Larger primary tumor size, hormone receptor negativity, HER2 positivity, higher nuclear grade, and number of pathologic nodal metastases were significant variables associated with HOXB9 expression. Notably, 12 (92.3%) of 13 triple-negative breast cancer cases showed HOXB9 expression. Disease-free survival and overall survival were significantly different between the HOXB9-positive and HOXB9-negative groups (hazard ratio 20.714, P?=?0.001; and hazard ratio 9.206, P?=?0.003, respectively). Multivariate analysis indicated that HOXB9 expression was the only independent prognostic factor for disease-free survival (hazard ratio 15.532, P?=?0.009). HOXB9-positive tumors showed a significant increase in the number of vasculature and the Ki-67 ratio compared with HOXB9-negative tumors.

Conclusions

HOXB9 expression, which promotes tumor proliferation and angiogenesis, is a significant prognostic factor in breast cancer.     

Reliability of cervical lordosis and global sagittal spinal balance measurements in adolescent idiopathic scoliosis

Study design

Radiological reproducibility study.

Purpose

To assess intra and interobserver reliability of radiographic measurements for global sagittal balance parameters and sagittal spine curves, including cervical spine.

Summary of background data

Sagittal spine balance in adolescent idiopathic scoliosis (AIS) is a main issue and many studies have been reported, showing that coronal and sagittal deformities often involve sagittal cervical unbalance. Global sagittal balance aims to obtain a horizontal gaze and gravity line at top of hips when subject is in a static position, involving adjustment of each spine curvature in the sagittal plane. To our knowledge, no study did use a methodologically validated imaging analysis tool able to appreciate sagittal spine contours and distances in AIS and especially in the cervical region.

Methods

Lateral full-spine low-dose EOS radiographs were performed in 75 patients divided in three groups (control subjects, AIS, operated AIS). Three observers digitally analyzed twice each radiograph and 11 sagittal measures were collected for each image. Reliability was assessed calculating intraobserver Pearson’s r correlation coefficient, interobserver intra-class correlation coefficient (ICC) completed with a two-by-two Bland–Altman plot analysis.

Results

This measurement method has shown excellent intra and interobserver reliability in all parameters, sagittal curvatures, pelvic parameters and global sagittal balance.

Conclusions

This study validated a simple and efficient tool in AIS sagittal contour analysis. It defined new relevant landmarks allowing to characterize cervical segmental curvatures and cervical involvement in global balance.     

Development and assessment of a digital X-ray software tool to determine vertebral rotation in adolescent idiopathic scoliosis

Background Context

The amount of vertebral rotation in the axial plane is of key importance in the prognosis and treatment of adolescent idiopathic scoliosis (AIS). Current methods to determine vertebral rotation are either designed for use in analogue plain radiographs and not useful in digital images, or lack measurement precision and are therefore less suitable for the follow-up of rotation in AIS patients.

Abnormal rib count in scoliosis surgery: impact on the reporting of spinal fusion levels

Purpose

Variation in rib numbering has been noted in adolescent idiopathic scoliosis (AIS), but its effect on the reporting of fusion levels has not been studied. We hypothesized that vertebral numbering variations can lead to differing documentation of fusion levels.

Methods

We examined the radiographs of 161 surgical AIS patients and 179 control patients without scoliosis. For AIS patients, the operative report of fusion levels was compared to conventional vertebral labeling from the first thoracic level and proceeding caudal. We defined normal counts as 12 thoracic (rib-bearing) and five lumbar (non-rib-bearing) vertebrae. We compared our counts with data from 181 anatomic specimens.

Results

Among AIS patients, 22 (14 %) had an abnormal number of ribs and 29 (18 %) had either abnormal rib or lumbar count. In 12/29 (41 %) patients, the operative report differed from conventional labeling by one level, versus 3/132 (2 %) patients with normal numbering (p < 0.001). However, there were no cases seen of wrong fusion levels based on curve pattern. Among controls, 11 % had abnormal rib count (p = 0.41) compared to the rate in AIS. Anatomic specimen data did not differ in abnormal rib count (p = 1.0) or thoracolumbar pattern (p = 0.59).

Conclusions

The rate of numerical variations in the thoracolumbar vertebrae of AIS patients is equivalent to that in the general population. When variations in rib count are present, differences in numbering levels can occur. In the treatment of scoliosis, no wrong fusion levels were noted. However, for both scoliosis patients and the general population, we suggest adherence to conventional labeling to enhance clarity.

     

Dextromethorphan inhibits osteoclast differentiation by suppressing RANKL-induced nuclear factor-κB activation

Summary

Dextromethorphan (DXM), a commonly used antitussive, is a dextrorotatory morphinan. Here, we report that DXM inhibits the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis and bone resorption by abrogating the activation of NF-κB signalling in vitro. Oral administration of DXM ameliorates ovariectomy (OVX)-induced osteoporosis in vivo.

Introduction

DXM was reported to possess anti-inflammatory properties through inhibition of the release of pro-inflammatory factors. However, the potential role and action mechanism of DXM on osteoclasts and osteoblasts remain unclear. In this study, in vitro and in vivo studies were performed to investigate the potential effects of DXM on osteoclastogenesis and OVX-induced bone loss.

Methods

Osteoclastogenesis was examined by the TRAP staining, pit resorption, TNF-α release, and CCR2 and CALCR gene expression. Osteoblast differentiation was analyzed by calcium deposition. Osteogenic and adipogenic genes were measured by real-time PCR. Signaling pathways were explored using Western blot. ICR mice were used in an OVX-induced osteoporosis model. Tibiae were measured by µCT and serum markers were examined with ELISA kits.

Results

DXM inhibited RANKL-induced osteoclastogenesis. DXM mainly inhibited osteoclastogenesis via abrogation of IKK-IκBα-NF-κB pathways. However, a higher dosage of DXM antagonized the differentiation of osteoblasts via the inhibition of osteogenic signals and increase of adipogenic signals. Oral administration of DXM (20 mg/kg/day) partially reduced trabecular bone loss in ovariectomized mice.

Conclusion

DXM inhibits osteoclast differentiation and activity by affecting NF-κB signaling. Therefore, DXM at suitable doses may have new therapeutic applications for the treatment of diseases associated with excessive osteoclastic activity.     

The genetic epidemiology of idiopathic scoliosis

Purpose

Idiopathic scoliosis is a complex developmental syndrome defined by an abnormal structural curvature of the spine. High treatment costs, chronic pain/discomfort, and the need for monitoring at-risk individuals contribute to the global healthcare burden of this musculoskeletal disease. Although many studies have endeavored to identify underlying genes, little progress has been made in understanding the etiopathogenesis. The objective of this comprehensive review was to summarize genetic associations/linkages with idiopathic scoliosis, as well as explore the strengths and weaknesses of each study, such that it may serve as a guide for the design and interpretation of future genetic studies in scoliosis.

Methods

We searched PubMed and Human Genome Epidemiology (HuGE) Navigator using the search terms “gene and scoliosis”. Linkage or association studies published in English and available full-text were further analyzed as regards results, experimental design, and statistical approach.

Results

We identified and analyzed 50 studies matching our criteria. These consisted of 34 candidate gene studies (6 linkage, 28 association) and 16 genome-wide studies [14 pedigree-based linkage, 2 genome-wide association studies (GWAS)]. Findings involved genes related to connective tissue structure, bone formation/metabolism, melatonin signaling pathways, puberty and growth, and axon guidance pathways. Variability in results between studies suggested ethnic and/or genetic heterogeneity.

Conclusions

The major difficulty in idiopathic scoliosis research is phenotypic and genetic heterogeneity. Genetic research was overrepresented by underpowered studies. The use of biological endophenotypes, as well as restricted clinical definitions, may help to partition variation and increase the power of studies to detect or confirm an effect.     

Effects of Pulsed Electromagnetic Fields on Human Osteoblastlike Cells (MG-63): A Pilot Study

Background

Although pulsed electromagnetic fields (PEMFs) are used to treat delayed unions and nonunions, their mechanisms of action are not completely clear. However, PEMFs are known to affect the expression of certain genes.

Questions/purposes

We asked (1) whether PEMFs affect gene expression in human osteoblastlike cells (MG63) in vitro, and (2) whether and to what extent stimulation by PEMFs induce cell proliferation and differentiation in MG-63 cultures.

Methods

We cultured two groups of MG63 cells. One group was treated with PEMFs for 18 hours whereas the second was maintained in the same culture condition without PEMFs (control). Gene expression was evaluated throughout cDNA microarray analysis containing 19,000 genes spanning a substantial fraction of the human genome.

Results

PEMFs induced the upregulation of important genes related to bone formation (HOXA10, AKT1), genes at the transductional level (CALM1, P2RX7), genes for cytoskeletal components (FN1, VCL), and collagenous (COL1A2) and noncollagenous (SPARC) matrix components. However, PEMF induced downregulation of genes related to the degradation of extracellular matrix (MMP-11, DUSP4).

Conclusions and Clinical Relevance

PEMFs appear to induce cell proliferation and differentiation. Furthermore, PEMFs promote extracellular matrix production and mineralization while decreasing matrix degradation and absorption. Our data suggest specific mechanisms of the observed clinical effect of PEMFs, and thus specific approaches for use in regenerative medicine.     

Wnt pathway genes in osteoporosis and osteoarthritis: differential expression and genetic association study

Summary

In comparison with hip fractures, increased expression of genes in the Wnt pathway and increased Wnt activity were found in bone samples and osteoblast cultures from patients with osteoarthritis, suggesting the involvement of this pathway in subchondral bone changes. No consistent differences were found in the genetic association study.

Introduction

This study aims to explore the allelic variations and expression of Wnt pathway genes in patients with osteoporosis and osteoarthritis.

Methods

The expression of 86 genes was studied in bone samples and osteoblast primary cultures from patients with hip fractures and hip or knee osteoarthritis. The Wnt-related activity was assessed by measuring AXIN2 and in transfection experiments. Fifty-five SNPs of the LRP5, LRP6, FRZB, and SOST genes were analyzed in 1,128 patients.

Results

Several genes were differentially expressed in bone tissue, with the lowest values usually found in hip fracture and the highest in knee osteoarthritis. Overall, seven genes were consistently upregulated both in tissue samples and in cell cultures from patients with knee osteoarthritis (BCL9, FZD5, DVL2, EP300, FRZB, LRP5, and TCF7L1). The increased expression of AXIN2 and experiments of transient transfection of osteoblasts with the TOP-Flash construct confirmed the activation of Wnt signaling. Three SNPs of the LRP5 gene and one in the LRP6 gene showed marginally significant differences in allelic frequencies across the patient groups, but they did not resist multiple-test adjustment.

Conclusions

Genes in the Wnt pathway are upregulated in the osteoarthritic bone, suggesting their involvement not only in cartilage distortion but also in subchondral bone changes.     

Reliability of assessing the coronal curvature of children with scoliosis by using ultrasound images

Purpose

To investigate the intra- and inter-observer reliability of the coronal curvature asymmetry of children with adolescent idiopathic scoliosis (AIS) using the center of lamina (COL) method on ultrasound (US) images.

Methods

A cadaver spinal column phantom which was manipulated to present 30 scoliotic curves of varying severity of scoliotic deformities was scanned using both the US and laser scanner (LS) systems. Three observers of varying experience and skill measured the coronal curvature using the Cobb method on the LS images and the COL method on the US images. All of the measurements were performed twice, with a 1-week interval to reduce memory bias. The intra-class correlation coefficient (ICC), the mean absolute differences (MAD), and the error index (EI) were calculated to determine the agreement on selecting the end vertebrae. In addition, five AIS subjects were scanned using the US system. One observer measured the coronal curvature on the US images twice, and the measurements were compared with the Cobb angle reported in the clinical records.

Results

In the phantom study, the COL method showed high intra- and inter-observer reliabilities, with all ICC values >0.88. The maximum MAD of the COL measurements between different sessions among all observers was <4.1°. The EI values of the US method had similar end-vertebra selections as the LS method. The results of the pilot study showed a high intra-reliability for the US measurements. The measured difference between the Cobb and COL methods was 0.7° ± 0.5°.

Conclusions

The COL method using US images appears to be a very reliable method for measuring the coronal curvature in AIS without the need to expose the patient to radiation.     

The metabolic basis of adolescent idiopathic scoliosis: 2011 report of the “metabolic” workgroup of the Fondation Yves Cotrel

Objective

The purpose of this review is to elucidate the metabolic processes involved in the pathogenesis of adolescent idiopathic scoliosis (AIS) in light of research by the present authors as well as current literature.

Methods

Pathogenetic mechanisms involved in AIS were modeled as (a) a form of neuromuscular scoliosis (in conjunction with an adverse mechanical environment such as bipedality), in which hormonal and other chemical factors act as regulators of skeletal muscle tone and function; (b) as a consequence of an abnormality in growth of the spinal column (in conjunction with an adverse mechanical environment such as bipedality), in which hormones and other chemical factors act as regulators of growth; and (c) as a mechanical failure of one side of the vertebral column due to a defect in trabecular formation or mineralization (in conjunction with an adverse mechanical environment such as bipedality); in which hormonal and other chemical factors act as regulators of bone formation, mineralization and/or resorption.

Results and conclusion

Current evidence supporting these models individually or in combination is discussed.     

Human osteoblast damage after antiseptic treatment

Purpose

Antiseptics are powerful medical agents used for wound treatment and decontamination and have a high potential for defeating joint infections in septic surgery. Both chlorhexidine and polyhexanide are frequently used in clinical practice and have a broad antimicrobial range, but their effect on human osteoblasts has not been sufficiently studied. Our objective was to investigate the toxic effects of polyhexanide and chlorhexidine on human osteoblasts in vitro to evaluate their clinical applicability in septic surgery.

Methods

We isolated and cultivated human osteoblasts in vitro and assayed the toxic effects of chlorhexidine 0.1 % and polyhexanide 0.04 %, concentrations commonly applied in clinical practice. Toxicity analysis was performed by visualisation of cell structure, lactate dehydrogenase (LDH) activity and evaluation of vital cells. Toxicity was evaluated by microscopic inspection of cell morphology, trypan blue staining and determination of LDH release.

Results

Damaged cell structure could be shown by microscopy. Both antiseptics promoted LDH activity after incubation with osteoblasts. The evaluation of vital osteoblasts showed a significant decrease of vital cells.

Conclusions

Both antiseptics induced significant cell death of osteoblasts at optimum exposure. We therefore recommend cautious use of polyhexanide and chlorhexidine in septic surgery to avoid severe osteoblast toxicity.     

A histomorphometric study of the cancellous spinal process bone in adolescent idiopathic scoliosis

Purpose

Adolescent idiopathic scoliosis (AIS) is a three-dimensional deformity with increased risk of osteopenia of unknown etiology. This study examined the dynamic histomorphometry of AIS patients to gain insight into the underlying pathogenesis of bone metabolism changes in AIS.

Methods

Bone histomorphometry of the spinous process of the 12th thoracic vertebra was analyzed in 33 AIS patients and compared to age-matched normative data. Patients were classified into bone turnover subgroups, based on bone formation rate.

Results

Bone volume was subnormal in 67% of AIS patients, but normal in 33%. Bone turnover was high in 76% of the patients, normal in 9%, and low in 15%. Compared to those in the low-turnover group, the high-turnover group patients were taller and had higher TRAP5b values.

Conclusions

Bone histomorphometry indicated that bone fragility and abnormal bone turnover were common in AIS patients. These abnormalities might contribute to the poor bone status and etiology in AIS.

     

Association between the ESR1 -351A>G single nucleotide polymorphism (rs9340799) and adolescent idiopathic scoliosis: a systematic review and meta-analysis

Purpose

A single nucleotide polymorphism in the promoter region of the estrogen receptor alpha gene (ESR1), rs9340799, has been linked with adolescent idiopathic scoliosis (AIS) in several association studies with limited sample size and inconsistent findings. A systematic review can provide a comprehensive appraisal of literature evidence and a meta-analysis can obtain a more precise estimate of any association. The purpose of the present study was to assess and synthesize the currently available evidence on the association between rs9340799 and AIS by conducting a systematic review and meta-analysis.

Methods

This review followed the Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines. PubMed (MEDLINE), EMBASE, Scopus and HuGE Literature Finder databases were systematically searched to identify relevant studies following a sensitive strategy. Summary odds ratios and corresponding 95 % confidence intervals (95 % CI) were estimated using the fixed-effect inverse variance model for allelic (G vs. A) and genotypic comparisons.

Results

Meta-analysis of four studies (n = 1,827 AIS cases and n = 1,253 controls) found a non-significant association between rs9340799 and AIS (allelic odds ratio 1.09, 95 % CI 0.96–1.23, p = 0.17).

Conclusions

When examined in isolation, the rs9340799 polymorphism does not appear to be a likely susceptibility variant for AIS predisposition. However, rs9340799 may be associated with AIS severity, progression and treatment; further investigation is necessary to confirm these potential associations.     

Lack of association between suppressor of cytokine signaling-3 gene polymorphism and susceptibility and curve severity of adolescent idiopathic scoliosis

Purpose

To explore whether the suppressor of cytokine signaling-3 (SOCS3) gene polymorphisms are associated with the susceptibility and abnormal growth pattern of adolescent idiopathic scoliosis (AIS).

Methods

Three hundred and ninety eight AIS girls aged 10–18 years old were enrolled, and 367 age-matched healthy girls were recruited as controls. Only patients who had Cobb angles larger than 20º were included in this study. Anthropometric parameters including body weight, height, and body mass index (BMI) were measured for AIS girls. Rs4969198 was selected as tagSNP to cover all of the related polymorphisms on SOCS3. Genotyping was performed using PCR-based Invader assay with the probe sets designed and synthesized by third wave. The genotyping results were read with an ABI PRISM7900HT sequence detection system (Applied Biosystems, Foster City, CA). A subgroup of 322 skeletally mature AIS patients who did not received bracing or any other conservative treatment previously were analyzed to define the contribution of rs4969168 on curve severity, body height, body weight, and BMI.

Results

Rs4969198 was successfully genotyped. No significant difference of genotype frequencies from the Hardy–Weinberg equilibrium (HWE) test was noted for the AIS patients or the normal controls. Neither the genotype nor the allele frequencies of rs49691968 were significantly different between the AIS patients and the normal controls. Rs4969168 was not found to be associated with age, curve severity of scoliosis, and body height. AIS patients with AA genotype had significantly higher body weight and BMI than the patients with AG and GG genotype (P = 0.014).

Conclusions

The SOCS3 gene polymorphisms are not associated with the occurrence of AIS, but the gene polymorphism (rs4969168) is associated with abnormal growth pattern of AIS, indicating that SOCS3 gene might be a disease-modifying gene of AIS.