Wenjie Gao

Association between Common Variants near LBX1 and Adolescent Idiopathic Scoliosis Replicated in the Chinese Han Population

Background Adolescent idiopathic scoliosis (AIS) is one of the most common spinal deformities found in adolescent populations. Recently, a genome-wide association study (GWAS) in a Japanese population indicated that three single nucleotide polymorphisms (SNPs), rs11190870, rs625039 and rs11598564, all located near the LBX1 gene, may be associated with AIS susceptibility [1]. This study suggests a novel AIS predisposition candidate gene and supports the hypothesis that somatosensory functional disorders could contribute to the pathogenesis of AIS. These findings warrant replication in other populations. Methodology/Principal Findings First, we conducted a case-control study consisting of 953 Chinese Han individuals from southern China (513 patients and 440 healthy controls), and the three SNPs were all found to be associated with AIS predisposition. The ORs were observed as 1.49 (95% CI 1.23–1.80, P = 5.09E-5), 1.70 (95% CI 1.42–2.04, P = 1.17E-8) and 1.52 (95% CI 1.27–1.83, P = 5.54E-6) for rs625039, rs11190870 and rs11598564, respectively. Second, a case-only study including a subgroup of AIS patients (N = 234) was performed to determine the effects of these variants on the severity of the condition. However, we did not find any association between these variants and the severity of curvature. Conclusion This study shows that the genetic variants near the LBX1 gene are associated with AIS susceptibility in Chinese Han population. It successfully replicates the results of the GWAS, which was performed in a Japanese population.

Endoplasmic reticulum stress-unfolding protein response-apoptosis cascade causes chondrodysplasia in a col2a1 p.Gly1170Ser mutated mouse model.

The collagen type II alpha 1 (COL2A1) mutation causes severe skeletal malformations, but the pathogenic mechanisms of how this occurs are unclear. To understand how this may happen, a col2a1 p.Gly1170Ser mutated mouse model was constructed and in homozygotes, the chondrodysplasia phenotype was observed. Misfolded procollagen was largely synthesized and retained in dilated endoplasmic reticulum and the endoplasmic reticulum stress (ERS)-unfolded protein response (UPR)-apoptosis cascade was activated. Apoptosis occurred prior to hypertrophy, prevented the formation of a hypertrophic zone, disrupted normal chondrogenic signaling pathways, and eventually caused chondrodysplasia. Heterozygotes had normal phenotypes and endoplasmic reticulum stress intensity was limited with no abnormal apoptosis detected. Our results suggest that earlier chondrocyte death was related to the ERS-UPR-apoptosis cascade and that this was the chief cause of chondrodysplaia. The col2a1 p.Gly1170Ser mutated mouse model offered a novel connection between misfolded collagen and skeletal malformation. Further investigation of this mouse mutant model can help us understand mechanisms of type II collagenopathies.

Normal leptin expression, lower adipogenic ability, decreased leptin receptor and hyposensitivity to Leptin in Adolescent Idiopathic Scoliosis.

Leptin has been suggested to play a role in the etiology of Adolescent Idiopathic Scoliosis (AIS), however, the leptin levels in AIS girls are still a discrepancy, and no in vitro study of leptin in AIS is reported. We took a series of case-control studies, trying to understand whether Leptin gene polymorphisms are involved in the etiology of the AIS or the change in leptin level is a secondary event, to assess the level of leptin receptor, and to evaluate the differences of response to leptin between AIS cases and controls. We screened all exons of Leptin gene in 45 cases and 45 controls and selected six tag SNPs to cover all the observed variations. Association analysis in 446 AIS patients and 550 healthy controls showed no association between the polymorphisms of Leptin gene and susceptibility/severity to AIS. Moreover, adipogenesis assay of bone mesenchymal stem cells (MSCs) suggested that the adipogenic ability of MSCs from AIS girls was lower than controls. After adjusting the differentiation rate, expressions of leptin and leptin receptor were similar between two groups. Meanwhile, osteogenesis assay of MSC showed the leptin level was similar after adjusting the differentiation rate, but the leptin receptor level was decreased in induced AIS osteoblasts. Immunocytochemistry and western blot analysis showed less leptin receptors expressed in AIS group. Furthermore, factorial designed studies with adipogenesis and osteogenesis revealed that the MSCs from patients have no response to leptin treatment. Our results suggested that Leptin gene variations are not associated with AIS and low serum leptin probably is a secondary outcome which may be related to the low capability of adipogenesis in AIS. The decreased leptin receptor levels may lead to the hyposensitivity to leptin. These findings implied that abnormal peripheral leptin signaling plays an important role in the pathological mechanism of AIS.

The relationship between concave angle of vertebral endplate and lumbar intervertebral disc degeneration.

Study Design. A retrospective study of magnetic resonance imaging of the lower lumbar spine. Objective. To describe the characteristics of the concave angle of vertebral endplate (CAVE) and study the association between CAVE and lumbar intervertebral disc degeneration (IVDD). Summary of Background Data. The vertebral endplate is responsible for transferring stress between disc and vertebral body, and its concavity is important in dispersing compression stress. However, the characteristics of CAVE and the relationship between CAVE and IVDD have not been investigated. Methods. Magnetic resonance images of the lower lumbar spine in 511 patients with low back pain were examined by 2 experienced spine surgeons. The grades of IVDD and lumbar disc herniation (LDH) were evaluated, several parameters including CAVE, height, and the sagittal diameter of vertebral body were measured, and the association between IVDD or LDH and CAVE was analyzed. Results. At L3–L4, L4–L5, and L5–S1, CAVE was smaller in the upper endplate (i.e., the inferior endplate of the superior vertebra) than in the lower endplate (i.e., the superior endplate of the inferior vertebra). There was no male/female difference in the size of CAVE in any of the segments. According to partial correlation analysis, CAVE was moderately related to IVDD, but no association between CAVE and LDH was found. Conclusion. When lumbar IVDD occurs, the CAVE increases and the endplate tends to flatten. The degree of flattening is related to the severity of the degeneration.

Melatonin enhances chondrogenic differentiation of human mesenchymal stem cells

Intramembranous ossification and endochondral ossification are two ways through which bone formation and fracture healing occur. Accumulating amounts of evidence suggests that melatonin affects osteoblast differentiation, but little is known about the effects of melatonin on the process of chondrogenic differentiation. In this study, the effects of melatonin on human mesenchymal stem cells (MSCs) undergoing chondrogenic differentiation were investigated. Cells were induced along chondrogenic differentiation via high‐density micromass culture in chondrogenic medium containing vehicle or 50 nm melatonin. Histological study and quantitative analysis of glycosaminoglycan (GAG) showed induced cartilage tissues to be larger and richer in GAG, collagen type II and collagen type X in the melatonin group than in the untreated controls. Real‐time RT‐PCR analysis demonstrated that melatonin treatment significantly up‐regulated the expression of the genes involved in chondrogenic differentiation, including aggrecan (ACAN), collagen type II (COL2A1), collagen type X (COL10A1), SRY (sex‐determining region Y)‐box 9 (SOX9), runt‐related transcription factor 2 (RUNX2) and the potent inducer of chondrogenic differentiation, bone morphogenetic protein 2 (BMP2). And the expression of melatonin membrane receptors (MT) MT1 and MT2 were detected in the chondrogenic‐induced‐MSCs by immunofluorescence staining. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to increase the size and GAG synthesis of the induced cartilage tissues, as well as to completely reverse the effect of melatonin on the gene expression of ACAN, COL2A1, COL10A1, SOX9 and BMP2 after 7 days of differentiation. These findings demonstrate that melatonin enhances chondrogenic differentiation of human MSCs at least partially through melatonin receptors.

The security analysis of transpedicular screw fixation in the lower cervical spine and a case report.

Study Design. This study evaluated the anatomical parameters of the lower cervical spine by imaging methods and reported a case. Objective. To explain low neurovascular injury rate and provide some suggestions in cervical transpedicular screw fixation (CTSF). Summary of Background Data. Because of anatomical complexity and possible severe complications, application of CTSF was limited. However, recent studies have indicated that although cervical pedicular screw perforations may happen, severe complications seldom occur. Methods. In 20 patients, several anatomical parameters were obtained on computed tomographic angiography (CTA) images of C3–C6, including the inner diameter of vertebral artery (d), the maximal width (A) and height (B) of the transverse foramen, the shortest distance between vertebral artery and cervical pedicle (h), and the angle between the longitudinal axis of pedicle and the hypothetical screw that just touches the vertebral artery (largest safe angle, LSA). Another 35 patients were chosen to measure the shortest distance between cervical pedicle and cervical spinal cord (H) and the angle between the longitudinal axis of pedicle and the hypothetical screw that just touches the cervical spinal cord (smallest angle, SA) on magnetic resonance imaging (MRI) of C4–C7. Results. Between the left and the right sides, there was no statistically significant difference for d, A, B, h, and LSA at C3–C6 or H and SA at C4–C7. d, h, and H were, respectively, 3.97 ± 0.65 mm, 0.89 ± 0.44 mm, and 6.56 ± 2.10 mm, and there was no statistically significant difference among C3–C6 for d and h or among C4–C7 for H. LSA at C6 was larger than that at C3, C4, and C5; it increased from C5 to C7. Conclusion. There was an “escaping space” for the vertebral artery and spinal cord. CTSF in the superior part of pedicle was relatively safer from accidental perforation of the vertebral artery than CTSF in the inferior part, and in C6 and C7, CTSF was safer from this injury than in C3, C4, and C5.

Melatonin‐mediated miR‐526b‐3p and miR‐590‐5p upregulation promotes chondrogenic differentiation of human mesenchymal stem cells

Bone marrow‐derived mesenchymal stem cells (BMSCs), with inherent chondrogenic differentiation potential appear to be ideally suited for therapeutic use in cartilage regeneration. Accumulating evidence has demonstrated that melatonin can promote chondrogenic differentiation in human BMSCs. However, little is known about the mechanism. MicroRNAs (miRNAs) have been shown to regulate the differentiation of BMSCs, but their roles in melatonin‐promoted chondrogenic differentiation have not been characterized. Here, we demonstrate that melatonin promoted chondrogenic differentiation of human BMSCs via upregulation of miR‐526b‐3p and miR‐590‐5p. Mechanistically, the elevated miR‐526b‐3p and miR‐590‐5p enhanced SMAD1 phosphorylation by targeting SMAD7. Additionally, administration of miR‐526b‐3p mimics or miR‐590‐5p mimics successfully promoted the chondrogenic differentiation of human BMSCs. Collectively, our study suggests that modification of BMSCs using melatonin or miRNA transduction could be an effective therapy for cartilage damage and degeneration.

Rare coding variants in MAPK7 predispose to adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis (AIS) is a complex genetic disorder characterized by three‐dimensional spinal curvatures, affecting 2%–3% of school age children, yet the causes underlying AIS are not well understood. Here, we first conducted a whole‐exome sequencing and linkage analysis on a three‐generation Chinese family with autosomal‐dominant (AD) AIS, and then performed targeted sequencing in a discovery cohort comprising 20 AD AIS families and 86 simplex patients, and finally identified three disease‐associated missense variants (c.886G> A, c.1943C> T, and c.1760C> T) in the MAPK7 gene (encoding mitogen‐activated protein kinase 7). Genotyping of the three rare variants in a Chinese replication cohort comprising 1,038 simplex patients and 1,841 controls showed that their combined allele frequency was significantly over‐represented in patients as compared with controls (2.0% [41/2,076] vs. 0.7% [27/3,682]; odds ratio = 2.7; P = 2.8 × 10−5). In vitro, we demonstrated that the three MAPK7 mutants disrupted nuclear translocation in cellular models, which is necessary for the normal function of MAPK7. In vivo, we also conducted CRISPR/Cas9‐mediated deletion of mapk7 in zebrafish recapitulating the characteristic phenotype of idiopathic scoliosis. Taken together, our findings suggest that rare coding variants in MAPK7 predispose to AIS, providing clues to understanding the mechanisms of AIS.