ABSTRACT
PURPOSE: This study aimed to undertake a multidisciplinary characterization of the phenotype associated with SOX11 variants. METHODS: Individuals with protein altering variants in SOX11 were identified through exome and genome sequencing and international data sharing. Deep clinical phenotyping was undertaken by referring clinicians. Blood DNA methylation was assessed using Infinium MethylationEPIC array. The expression pattern of SOX11 in developing human brain was defined using RNAscope. RESULTS: We reported 38 new patients with SOX11 variants. Idiopathic hypogonadotropic hypogonadism was confirmed as a feature of SOX11 syndrome. A distinctive pattern of blood DNA methylation was identified in SOX11 syndrome, separating SOX11 syndrome from other BAFopathies. CONCLUSION: SOX11 syndrome is a distinct clinical entity with characteristic clinical features and episignature differentiating it from BAFopathies.
Subject(s)
DNA Methylation , Hypogonadism , Klinefelter Syndrome , Neurodevelopmental Disorders , SOXC Transcription Factors , DNA Methylation/genetics , Humans , Hypogonadism/genetics , Klinefelter Syndrome/genetics , Neurodevelopmental Disorders/genetics , Phenotype , SOXC Transcription Factors/genetics , Exome SequencingABSTRACT
Adolescent idiopathic scoliosis (AIS) is the most common musculoskeletal disorder of childhood development. The genetic architecture of AIS is complex, and the great majority of risk factors are undiscovered. To identify new AIS susceptibility loci, we conducted the first genome-wide meta-analysis of AIS genome-wide association studies, including 7956 cases and 88 459 controls from 3 ancestral groups. Three novel loci that surpassed genome-wide significance were uncovered in intragenic regions of the CDH13 (P-value_rs4513093 = 1.7E-15), ABO (P-value_ rs687621 = 7.3E-10) and SOX6 (P-value_rs1455114 = 2.98E-08) genes. Restricting the analysis to females improved the associations at multiple loci, most notably with variants within CDH13 despite the reduction in sample size. Genome-wide gene-functional enrichment analysis identified significant perturbation of pathways involving cartilage and connective tissue development. Expression of both SOX6 and CDH13 was detected in cartilage chondrocytes and chromatin immunoprecipitation sequencing experiments in that tissue revealed multiple HeK27ac-positive peaks overlapping associated loci. Our results further define the genetic architecture of AIS and highlight the importance of vertebral cartilage development in its pathogenesis.
Subject(s)
ABO Blood-Group System/genetics , Cadherins/genetics , Musculoskeletal Diseases/genetics , SOXD Transcription Factors/genetics , Scoliosis/genetics , Adolescent , Child , Ethnicity/genetics , Female , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Male , Musculoskeletal Diseases/physiopathology , Polymorphism, Single Nucleotide/genetics , Scoliosis/physiopathology , Young AdultABSTRACT
BACKGROUND: Congenital scoliosis (CS) is a common vertebral malformation. Spondylocostal dysostosis (SCD) is a rare skeletal dysplasia characterised by multiple vertebral malformations and rib anomalies. In a previous study, a compound heterozygosity for a null mutation and a risk haplotype composed by three single-nucleotide polymorphisms in TBX6 have been reported as a disease-causing model of CS. Another study identified bi-allelic missense variants in a SCD patient. The purpose of our study is to identify TBX6 variants in CS and SCD and examine their pathogenicity. METHODS: We recruited 200 patients with CS or SCD and investigated TBX6 variants. We evaluated the pathogenicity of the variants by in silico prediction and in vitro experiments. RESULTS: We identified five 16p11.2 deletions, one splice-site variant and five missense variants in 10 patients. In vitro functional assays for missense variants identified in the previous and present studies demonstrated that most of the variants caused abnormal localisation of TBX6 proteins. We confirmed mislocalisation of TBX6 proteins in presomitic mesoderm cells induced from SCD patient-derived iPS cells. In induced cells, we found decreased mRNA expressions of TBX6 and its downstream genes were involved in somite formation. All CS patients with missense variants had the risk haplotype in the opposite allele, while a SCD patient with bi-allelic missense variants did not have the haplotype. CONCLUSIONS: Our study suggests that bi-allelic loss of function variants of TBX6 cause a spectrum of phenotypes including CS and SCD, depending on the severity of the loss of TBX6 function.
Subject(s)
Abnormalities, Multiple/diagnosis , Abnormalities, Multiple/genetics , Alleles , Hernia, Diaphragmatic/diagnosis , Hernia, Diaphragmatic/genetics , Loss of Function Mutation , Scoliosis/congenital , Scoliosis/diagnosis , Spine/abnormalities , T-Box Domain Proteins/genetics , Computational Biology/methods , Gene Expression , Genetic Association Studies , Genetic Predisposition to Disease , Genotype , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mutation, MissenseABSTRACT
Adolescent idiopathic scoliosis (AIS) is a common spinal deformity affecting millions of children. Since treatment and prognosis of AIS depend on curve progression, identifying factors related to AIS curve progression is important in its management. Although several genetic loci for AIS occurrence are reported, no locus for curve progression has been identified. To identify genes associated with AIS progression, we conducted a genome-wide association study followed by a replication study using a total of 2,543 AIS subjects who were evaluated for the curve progression. We identified a significantly associated locus on chromosome 11q14.1 (P = 1.98 × 10-9, odds ratio = 1.56). In silico and in vitro analyses identified a functional variant, rs35333564 in MIR4300HG, the host gene of a microRNA, MIR4300. The genomic region containing rs35333564 had enhancer activity, which was decreased in its risk allele. Our data suggest that decrease of MIR4300 is related to AIS progression.
Subject(s)
MicroRNAs/genetics , Scoliosis/genetics , Adolescent , Alleles , Asian People/genetics , Disease Progression , Female , Gene Frequency , Genetic Predisposition to Disease , Genome-Wide Association Study , Humans , Japan , Male , MicroRNAs/metabolism , Odds Ratio , Polymorphism, Single Nucleotide , Risk Factors , Scoliosis/metabolismABSTRACT
Ossification of the posterior longitudinal ligament of the spine (OPLL) is a common spinal disorder that results from ectopic ossification of the posterior longitudinal ligament and causes intractable myelopathy and radiculopathy. In a previous genome-wide association study (GWAS), we found six loci associated with OPLL; however, susceptibility genes in these loci have not been identified yet. Here, we examined one of the GWAS loci and identified RSPO2 (encoding R-spondin 2) as a susceptibility gene for OPLL. R-spondin 2 is a secreted agonist of canonical Wnt-ß-catenin signaling. RSPO2 was decreased in the early stage of chondrocyte differentiation. R-spondin 2 inhibited expression of genes encoding early chondrocyte differentiation markers by activating Wnt-ß-catenin signaling. rs374810, the most significantly associated SNP in the GWAS locus in chromosomal region 8q23.1 was located in the chondrocyte promoter region of RSPO2. A transcription factor, CCAAT-enhancer-binding protein ß (C/EBPß), specifically bound to the RSPO2 core promoter region containing rs374810 and increased RSPO2 expression. The risk allele of rs374810 affected the binding of the promoter with C/EBPß and decreased the RSPO2 transcription in vitro and in vivo. Our genetic and functional data indicate that RSPO2 is a susceptibility gene for OPLL.
Subject(s)
Genetic Predisposition to Disease/genetics , Intercellular Signaling Peptides and Proteins/genetics , Ossification of Posterior Longitudinal Ligament/genetics , Cell Line , Humans , Polymorphism, Single Nucleotide/genetics , Wnt Signaling Pathway , beta Catenin/metabolismABSTRACT
Adolescent idiopathic scoliosis (AIS) is the most common type of scoliosis. Controlling its curve progression is the most important clinical task. Although recent genome-wide association studies (GWASs) identified several susceptibility loci associated with the development of AIS, the etiology of curve progression has been still unknown. Our previous GWAS has identified that rs12946942 showed significant association with severe AIS. To confirm the association, we conducted an international meta-analysis using four cohorts with different ethnicity. We analyzed 2272 severe AIS cases and 13,859 controls in total, and found the replication of significant association of rs12946942 (combined P = 7.23×10-13; odds ratio = 1.36, 95% confidence interval = 1.25-1.49). In silico analyses suggested that SOX9 is the most likely susceptibility gene for AIS curve progression in the locus.
Subject(s)
Genetic Predisposition to Disease , Genome-Wide Association Study , Polymorphism, Genetic , SOX9 Transcription Factor/genetics , Scoliosis/ethnology , Scoliosis/genetics , Adolescent , Female , Humans , MaleABSTRACT
Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish.
Subject(s)
Homeodomain Proteins/genetics , Morphogenesis/genetics , Scoliosis/genetics , Transcription Factors/genetics , Wnt Proteins/genetics , Zebrafish Proteins/genetics , Adolescent , Animals , Cell Polarity/genetics , Gene Expression Regulation, Developmental , HEK293 Cells , Homeodomain Proteins/biosynthesis , Humans , Polymorphism, Single Nucleotide , Scoliosis/pathology , Transcription Factors/biosynthesis , Wnt Proteins/biosynthesis , Wnt Signaling Pathway/genetics , Wnt-5a Protein , Zebrafish , Zebrafish Proteins/biosynthesisABSTRACT
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.
Subject(s)
Chromosomes, Human, Pair 9/genetics , DNA-Binding Proteins/genetics , Genetic Predisposition to Disease , Phenotype , Polymorphism, Single Nucleotide/genetics , Scoliosis/genetics , Adolescent , Animals , China , DNA-Binding Proteins/metabolism , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/pathology , Genome-Wide Association Study , Humans , Japan , Luciferases , Odds Ratio , Scoliosis/pathology , YY1 Transcription Factor/metabolism , ZebrafishABSTRACT
Congenital scoliosis (CS) occurs as a result of vertebral malformations and has an incidence of 0.5-1/1,000 births. Recently, TBX6 on chromosome 16p11.2 was reported as a disease gene for CS; about 10% of Chinese CS patients were compound heterozygotes for rare null mutations and a common haplotype defined by three SNPs in TBX6. All patients had hemivertebrae. We recruited 94 Japanese CS patients, investigated the TBX6 locus for both mutations and the risk haplotype, examined transcriptional activities of mutant TBX6 in vitro, and evaluated clinical and radiographic features. We identified TBX6 null mutations in nine patients, including a missense mutation that had a loss of function in vitro. All had the risk haplotype in the opposite allele. One of the mutations showed dominant negative effect. Although all Chinese patients had one or more hemivertebrae, two Japanese patients did not have hemivertebra. The compound heterozygosity of null mutations and the common risk haplotype in TBX6 also causes CS in Japanese patients with similar incidence. Hemivertebra was not a specific type of spinal malformation in TBX6-associated CS (TACS). A heterozygous TBX6 loss-of-function mutation has been reported in a family with autosomal-dominant spondylocostal dysostosis, but it may represent a spectrum of the same disease with TACS.
Subject(s)
Congenital Abnormalities/genetics , Haplotypes , Heterozygote , Loss of Function Mutation , Scoliosis/genetics , T-Box Domain Proteins/genetics , Adolescent , Child , Child, Preschool , Chromosome Deletion , Chromosomes, Human, Pair 16 , Congenital Abnormalities/diagnosis , DNA Mutational Analysis , Female , Humans , Male , Pedigree , Phenotype , Radiography , Scoliosis/diagnosisABSTRACT
BACKGROUND: SOX11 is a transcription factor proposed to play a role in brain development. The relevance of SOX11 to human developmental disorders was suggested by a recent report of SOX11 mutations in two patients with Coffin-Siris syndrome. Here we further investigate the role of SOX11 variants in neurodevelopmental disorders. METHODS: We used array based comparative genomic hybridisation and trio exome sequencing to identify children with intellectual disability who have deletions or de novo point mutations disrupting SOX11. The pathogenicity of the SOX11 mutations was assessed using an in vitro gene expression reporter system. Loss-of-function experiments were performed in xenopus by knockdown of Sox11 expression. RESULTS: We identified seven individuals with chromosome 2p25 deletions involving SOX11. Trio exome sequencing identified three de novo SOX11 variants, two missense (p.K50N; p.P120H) and one nonsense (p.C29*). The biological consequences of the missense mutations were assessed using an in vitro gene expression system. These individuals had microcephaly, developmental delay and shared dysmorphic features compatible with mild Coffin-Siris syndrome. To further investigate the function of SOX11, we knocked down the orthologous gene in xenopus. Morphants had significant reduction in head size compared with controls. This suggests that SOX11 loss of function can be associated with microcephaly. CONCLUSIONS: We thus propose that SOX11 deletion or mutation can present with a Coffin-Siris phenotype.
Subject(s)
Abnormalities, Multiple/genetics , Face/abnormalities , Hand Deformities, Congenital/genetics , Intellectual Disability/genetics , Micrognathism/genetics , Neck/abnormalities , Neurodevelopmental Disorders/genetics , SOXC Transcription Factors/genetics , Sequence Deletion , Abnormalities, Multiple/physiopathology , Adolescent , Adult , Animals , Child , Child, Preschool , Face/physiopathology , Female , Gene Knockdown Techniques , Hand Deformities, Congenital/physiopathology , Humans , Intellectual Disability/physiopathology , Male , Microcephaly , Micrognathism/physiopathology , Neck/physiopathology , Neurodevelopmental Disorders/physiopathology , XenopusABSTRACT
Adolescent idiopathic scoliosis(AIS)is a polygenic disease. Genome-wide association studies(GWASs)have been performed for a lot of polygenic diseases. For AIS, we conducted GWAS and identified the first AIS locus near LBX1. After the discovery, we have extended our study by increasing the numbers of subjects and SNPs. In total, our Japanese GWAS has identified four susceptibility genes. GWASs for AIS have also been performed in the USA and China, which identified one and three susceptibility genes, respectively. Here we review GWASs in Japan and abroad and functional analysis to clarify the pathomechanism of AIS.
Subject(s)
Scoliosis/genetics , Adolescent , Animals , Chromosomes, Human, Pair 9 , Genetic Predisposition to Disease , Genome, Human , Genome-Wide Association Study , Humans , Polymorphism, Single Nucleotide , Scoliosis/epidemiologyABSTRACT
BACKGROUND: Adolescent idiopathic scoliosis (AIS) is a common rotational deformity of the spine that presents in children worldwide, yet its etiology is poorly understood. Recent genome-wide association studies (GWAS) have identified a few candidate risk loci. One locus near the chromosome 10q24.31 LBX1 gene (OMIM #604255) was originally identified by a GWAS of Japanese subjects and replicated in additional Asian populations. To extend this result, and to create larger AIS cohorts for the purpose of large-scale meta-analyses in multiple ethnicities, we formed a collaborative group called the International Consortium for Scoliosis Genetics (ICSG). METHODS: Here, we report the first ICSG study, a meta-analysis of the LBX1 locus in six Asian and three non-Asian cohorts. RESULTS: We find significant evidence for association of this locus with AIS susceptibility in all nine cohorts. Results for seven cohorts containing both genders yielded P=1.22×10-43 for rs11190870, and P=2.94×10-48 for females in all nine cohorts. Comparing the regional haplotype structures for three populations, we refined the boundaries of association to a â¼25 kb block encompassing the LBX1 gene. The LBX1 protein, a homeobox transcription factor that is orthologous to the Drosophila ladybird late gene, is involved in proper migration of muscle precursor cells, specification of cardiac neural crest cells, and neuronal determination in developing neural tubes. CONCLUSIONS: Our results firmly establish the LBX1 region as the first major susceptibility locus for AIS in Asian and non-Hispanic white groups, and provide a platform for larger studies in additional ancestral groups.
Subject(s)
Asian People/genetics , Homeodomain Proteins/genetics , Scoliosis/genetics , Transcription Factors/genetics , Adolescent , Female , Genome-Wide Association Study , Humans , Linkage Disequilibrium , Male , Polymorphism, Single NucleotideABSTRACT
BACKGROUND: Osteoporotic fracture (OF) as a clinical endpoint is a major complication of osteoporosis. To screen for OF susceptibility genes, we performed a genome-wide association study and carried out de novo replication analysis of an East Asian population. METHODS: Association was tested using a logistic regression analysis. A meta-analysis was performed on the combined results using effect size and standard errors estimated for each study. RESULTS: In a combined meta-analysis of a discovery cohort (288 cases and 1139 controls), three hospital based sets in replication stage I (462 cases and 1745 controls), and an independent ethnic group in replication stage II (369 cases and 560 for controls), we identified a new locus associated with OF (rs784288 in the MECOM gene) that showed genome-wide significance (p=3.59×10(-8); OR 1.39). RNA interference revealed that a MECOM knockdown suppresses osteoclastogenesis. CONCLUSIONS: Our findings provide new insights into the genetic architecture underlying OF in East Asians.
Subject(s)
DNA-Binding Proteins/genetics , Osteoporosis/genetics , Osteoporotic Fractures/genetics , Proto-Oncogenes/genetics , Quantitative Trait Loci/genetics , Transcription Factors/genetics , Aged , Case-Control Studies , Gene Expression Regulation , Gene Knockdown Techniques , Genetic Predisposition to Disease , Genome-Wide Association Study , Genotype , Humans , MDS1 and EVI1 Complex Locus Protein , Middle Aged , Osteogenesis/genetics , Osteoporosis/pathology , Osteoporotic Fractures/pathology , Polymorphism, Single NucleotideABSTRACT
Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-beta-mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-beta activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-beta. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-beta activity and disease, suggesting new therapeutic strategies for osteoarthritis.
Subject(s)
Chondrogenesis/genetics , Extracellular Matrix Proteins/genetics , Glycoproteins/genetics , Osteoarthritis/genetics , Polymorphism, Genetic , Aggrecans , Aspartic Acid/genetics , Carrier Proteins , Chromosome Mapping , Disease Susceptibility , Humans , In Vitro Techniques , Lectins, C-Type , Minisatellite Repeats , Molecular Sequence Data , Osteoarthritis, Hip/genetics , Osteoarthritis, Knee/genetics , Proteoglycans/genetics , Transforming Growth Factor beta/antagonists & inhibitorsABSTRACT
We previously reported 2 osteoporosis-susceptibility genes--formiminotransferase N-terminal sub-domain containing gene (FONG) and thrombospondin, type 1, domain-containing 7A (THSD7A)--in which we identified two common single-nucleotide polymorphisms, rs7605378 (FONG) and rs12673692 (THSD7A). The former was associated with a predisposition to osteoporosis and the latter with bone mineral density. To further elucidate the importance of these polymorphisms in the pathogenesis of osteoporosis, we examined their association with the incidence of vertebral fracture. DNA extracted from the renal cortex of 2427 consecutive Japanese autopsies (1331 men, mean age: 79 years; 1096 women, mean age: 82 years) were examined in this study. The presence or absence of vertebral fracture during each subject's lifetime was determined by a thorough examination of the clinical records, as well as autopsy reports. After adjustments for sex and age at autopsy, logistic regression analysis revealed that homozygotes for the risk alleles of rs7605378 (A-allele) or rs12673629 (A-allele) possess an increased risk of vertebral fracture. The subjects simultaneously homozygous for both the risk alleles of rs7605378 (AA genotype) and rs12673629 (AA genotype) showed significantly higher risk of vertebral fracture (odds ratio 2.401, 95% confidence interval 1.305-4.416, P = 0.0048) than those who had at least one non-risk allele of either rs7605378 (AC/CC genotypes) or rs12673629 (AG/GG genotypes). The results suggest that Japanese subjects homozygous for the risk alleles of rs7605378 and rs12673629 have a higher risk of vertebral fracture.
Subject(s)
Glutamate Formimidoyltransferase/genetics , Hydroxymethyl and Formyl Transferases/genetics , Spinal Fractures/genetics , Thrombospondins/genetics , Aged , Aged, 80 and over , Autopsy , Female , Genotype , Glutamate Formimidoyltransferase/chemistry , Humans , MaleABSTRACT
A study was conducted to validate the most significant single nucleotide polymorphism (SNP) from a genome-wide association study of Japanese adolescent idiopathic scoliosis (AIS) patients in an independent southern Chinese population. In total, 300 AIS patients fulfilled the clinical criteria and 788 controls with MRI scans of the spine were included in the replication study. We employed case-control analysis to study the association of SNP rs11190870 near LBX1 (ladybird homeobox 1) with AIS in a southern Chinese population. The results suggest that SNP rs11190870 is significantly associated with AIS (P=9.1 × 10(-10); odds ratio=1.85; 95% confidence interval=1.52-2.25). The results of this study confirm that SNP rs11190870 is associated with AIS.
Subject(s)
Asian People/genetics , Homeodomain Proteins/genetics , Polymorphism, Single Nucleotide , Scoliosis/genetics , Transcription Factors/genetics , Adolescent , Case-Control Studies , Child , Female , Gene Frequency , Genetic Predisposition to Disease , Genetics, Population , Genome-Wide Association Study , Humans , Magnetic Resonance Imaging , Male , Odds Ratio , Sex Factors , Young AdultABSTRACT
Bone mineral density (BMD) is one of the major determinants of risk for osteoporotic fracture. Multiple studies reveal that peak bone mass is under strong genetic influence. One of the major susceptibility loci for peak spine BMD has been mapped to chromosome 1q21-q23 in the Caucasian population. We have previously replicated this finding in Southern Chinese pedigrees and detected a maximum multipoint log of odds (LOD) score of 2.36 in this region. To further fine-map this region, 380 single-nucleotide polymorphic (SNP) markers were genotyped in 610 sibpairs from 231 families. Several markers were identified in the association analysis as important candidates underlying BMD variation. Among them, successful replication was demonstrated for SNPs in pre-B-cell leukemia homeobox 1 (PBX1) gene in two other unrelated case-control cohorts. The functional role of PBX1 in bone metabolism was examined in vitro using human bone-derived cells (HBDC) and murine MC3T3-E1 pre-osteoblasts. PBX1 mRNA was constitutively expressed in both HBDC and MC3T3-E1 cells. Immunostaining revealed that PBX1 is localized in the nucleus compartment. Silencing of PBX1 by RNAi in MC3T3-E1 cells decreased the expression of Runx2 and Osterix, the critical transcription factors for osteogenesis, but accelerated cell proliferation and bone nodule formation. Overall, our data suggest a genetic and functional association of PBX1 with BMD.
Subject(s)
Bone Density , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Osteoporosis/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Animals , Asian People/genetics , Case-Control Studies , Cell Line , China , Cohort Studies , Female , Humans , Japan , Male , Mice , Osteoblasts/metabolism , Osteoporosis/metabolism , Pedigree , Polymorphism, Single Nucleotide , Pre-B-Cell Leukemia Transcription Factor 1ABSTRACT
A significant association between a polymorphism in the D repeat of the gene encoding asporin and osteoarthritis, the most frequent of articular diseases, has been recently reported. The goal of the present study was to investigate the expression of this new class I small leucine-rich proteoglycan (SLRP) in human articular chondrocytes. First, we studied the modulation of asporin (ASPN) expression by cytokines by Western blot and reverse transcription-polymerase chain reaction. Interleukin-1ß and tumor necrosis factor-α downregulated ASPN, whereas transforming growth factor-ß1 (when incubated in a serum-free medium) upregulated it. Similarly to proinflammatory cytokines, chondrocyte dedifferentiation induced by a successive passages of cells was accompanied by a decreased asporin expression, whereas their redifferentiation by three-dimensional culture restored its expression. Finally, we found an important role of the transcription factor Sp1 in the regulation of ASPN expression. Sp1 ectopic expression increased ASPN mRNA level and promoter activity. In addition, using gene reporter assay and electrophoretic mobility shift assay, we showed that Sp1 mediated its effect through a region located between -473 and -140 bp upstream of the transcription start site in ASPN gene. In conclusion, this report is the first study on the regulation of asporin expression by different cytokines in human articular chondrocytes. Our data indicate that the expression of this gene is finely regulated in cartilage and suggest a major role of Sp1.
Subject(s)
Chondrocytes/metabolism , Extracellular Matrix Proteins/genetics , Gene Expression/genetics , Aged , Aged, 80 and over , Binding Sites/genetics , Blotting, Western , Cartilage, Articular/cytology , Cell Dedifferentiation/genetics , Cell Differentiation/genetics , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/drug effects , Down-Regulation/drug effects , Electrophoretic Mobility Shift Assay , Extracellular Matrix Proteins/metabolism , Gene Expression/drug effects , Humans , Interleukin-1beta/pharmacology , Middle Aged , Primary Cell Culture , Promoter Regions, Genetic/genetics , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Transfection , Transforming Growth Factor beta1/pharmacology , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Adolescent idiopathic scoliosis (AIS) is a common disease causing three-dimensional spinal deformity in as many as 3% of adolescents. Development of a method that can accurately predict the onset and progression of AIS is an immediate need for clinical practice. Because the heritability of AIS is estimated as high as 87.5% in twin studies, prediction of its onset and progression based on genetic data is a promising option. We show the usefulness of polygenic risk score (PRS) for the prediction of onset and progression of AIS. We used AIS genomewide association study (GWAS) data comprising 79,211 subjects in three cohorts and constructed a PRS based on association statistics in a discovery set including 31,999 female subjects. After calibration using a validation data set, we applied the PRS to a test data set. By integrating functional annotations showing heritability enrichment in the selection of variants, the PRS demonstrated an association with AIS susceptibility (p = 3.5 × 10-40 with area under the receiver-operating characteristic [AUROC] = 0.674, sensitivity = 0.644, and specificity = 0.622). The decile with the highest PRS showed an odds ratio of as high as 3.36 (p = 1.4 × 10-10 ) to develop AIS compared with the fifth in decile. The addition of a predictive model with only a single clinical parameter (body mass index) improved predictive ability for development of AIS (AUROC = 0.722, net reclassification improvement [NRI] 0.505 ± 0.054, p = 1.6 × 10-8 ), potentiating clinical use of the prediction model. Furthermore, we found the Cobb angle (CA), the severity measurement of AIS, to be a polygenic trait that showed a significant genetic correlation with AIS susceptibility (rg = 0.6, p = 3.0 × 10-4 ). The AIS PRS demonstrated a significant association with CA. These results indicate a shared polygenic architecture between onset and progression of AIS and the potential usefulness of PRS in clinical settings as a predictor to promote early intervention of AIS and avoid invasive surgery. © 2021 American Society for Bone and Mineral Research (ASBMR).
Subject(s)
Kyphosis , Scoliosis , Adolescent , Bone and Bones , Female , Genome-Wide Association Study , Humans , Risk Factors , Scoliosis/geneticsABSTRACT
Asporin is an extracellular matrix (ECM) protein that regulates cartilage matrix gene expression and cartilage formation by modulating the transforming growth factor-beta (TGF-beta) signaling pathway. Our previous studies have indicated that asporin binds to TGF-beta1 directly and inhibits TGF-beta1-mediated expression of cartilage matrix genes. However, it is still unknown how asporin interacts with TGF-beta1 and influences its activity. Using competition assays, we determined that amino acids 159-205 of asporin mediate its interaction with TGF-beta1 and effectively repress TGF-beta1-induced cartilage matrix gene expression. Asporin also has a binding ability to type II collagen in vitro, but its binding pattern is different from that of TGF-beta1. In contrast with previous in vivo findings, asporin did not affect the interaction between TGF-beta1 and the TGF-beta type II receptor (TbetaRII) by itself or in the presence of type II collagen in vitro. However, in the presence of heparin/heparan sulfate, asporin inhibits the interaction between TGF-beta and TbetaRII in vitro. These findings suggest that asporin is one of the important cartilage matrix proteins that binds to the ECM and TGF-beta1 and thereby modulates interactions between TGF-beta and its signaling receptors.