ABSTRACT
Our previous genome-wide association study (GWAS) for sagittal nonsyndromic craniosynostosis (sNCS) provided important insights into the genetics of midline CS. In this study, we performed a GWAS for a second midline NCS, metopic NCS (mNCS), using 215 non-Hispanic white case-parent triads. We identified six variants with genome-wide significance (P ≤ 5 × 10-8): rs781716 (P = 4.71 × 10-9; odds ratio [OR] = 2.44) intronic to SPRY3; rs6127972 (P = 4.41 × 10-8; OR = 2.17) intronic to BMP7; rs62590971 (P = 6.22 × 10-9; OR = 0.34), located ~ 155 kb upstream from TGIF2LX; and rs2522623, rs2573826, and rs2754857, all intronic to PCDH11X (P = 1.76 × 10-8, OR = 0.45; P = 3.31 × 10-8, OR = 0.45; P = 1.09 × 10-8, OR = 0.44, respectively). We performed a replication study of these variants using an independent non-Hispanic white sample of 194 unrelated mNCS cases and 333 unaffected controls; only the association for rs6127972 (P = 0.004, OR = 1.45; meta-analysis P = 1.27 × 10-8, OR = 1.74) was replicated. Our meta-analysis examining single nucleotide polymorphisms common to both our mNCS and sNCS studies showed the strongest association for rs6127972 (P = 1.16 × 10-6). Our imputation analysis identified a linkage disequilibrium block encompassing rs6127972, which contained an enhancer overlapping a CTCF transcription factor binding site (chr20:55,798,821-55,798,917) that was significantly hypomethylated in mesenchymal stem cells derived from fused metopic compared to open sutures from the same probands. This study provides additional insights into genetic factors in midline CS.
Subject(s)
Bone Morphogenetic Protein 7/genetics , Craniosynostoses/genetics , Genetic Variation , Polymorphism, Single Nucleotide/genetics , Alleles , DNA Methylation , Genes, Reporter , Genetic Predisposition to Disease , Genome-Wide Association Study , Genotype , Humans , Introns/genetics , Linkage Disequilibrium , Promoter Regions, Genetic/genetics , Risk FactorsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
PURPOSE: Enrichment of heterozygous missense and truncating SMAD6 variants was previously reported in nonsyndromic sagittal and metopic synostosis, and interaction of SMAD6 variants with a common polymorphism nearBMP2 (rs1884302) was proposed to contribute to inconsistent penetrance. We determined the occurrence of SMAD6 variants in all types of craniosynostosis, evaluated the impact of different missense variants on SMAD6 function, and tested independently whether rs1884302 genotype significantly modifies the phenotype. METHODS: We performed resequencing of SMAD6 in 795 unsolved patients with any type of craniosynostosis and genotyped rs1884302 in SMAD6-positive individuals and relatives. We examined the inhibitory activity and stability of SMAD6 missense variants. RESULTS: We found 18 (2.3%) different rare damaging SMAD6 variants, with the highest prevalence in metopic synostosis (5.8%) and an 18.3-fold enrichment of loss-of-function variants comparedwith gnomAD data (P < 10-7). Combined with eight additional variants, ≥20/26 were transmitted from an unaffected parent but rs1884302 genotype did not predict phenotype. CONCLUSION: Pathogenic SMAD6 variants substantially increase the risk of both nonsyndromic and syndromic presentations of craniosynostosis, especially metopic synostosis. Functional analysis is important to evaluate missense variants. Genotyping of rs1884302 is not clinically useful. Mechanisms to explain the remarkable diversity of phenotypes associated with SMAD6 variants remain obscure.
Subject(s)
Craniosynostoses , Craniosynostoses/genetics , Genotype , Humans , Mutation, Missense/genetics , Penetrance , Phenotype , Smad6 Protein/geneticsABSTRACT
Craniosynostosis presents either as a nonsyndromic congenital anomaly or as a finding in nearly 200 genetic syndromes. Our previous genome-wide association study of sagittal nonsyndromic craniosynostosis identified associations with variants downstream from BMP2 and intronic in BBS9. Because no coding variants in BMP2 were identified, we hypothesized that conserved non-coding regulatory elements may alter BMP2 expression. In order to identify and characterize noncoding regulatory elements near BMP2, two conserved noncoding regions near the associated region on chromosome 20 were tested for regulatory activity with a Renilla luciferase assay. For a 711 base pair noncoding fragment encompassing the most strongly associated variant, rs1884302, the luciferase assay showed that the risk allele (C) of rs1884302 drives higher expression of the reporter than the common allele (T). When this same DNA fragment was tested in zebrafish transgenesis studies, a strikingly different expression pattern of the green fluorescent reporter was observed depending on whether the transgenic fish had the risk (C) or the common (T) allele at rs1884302. The in vitro results suggest that altered BMP2 regulatory function at rs1884302 may contribute to the etiology of sagittal nonsyndromic craniosynostosis. The in vivo results indicate that differences in regulatory activity depend on the presence of a C or T allele at rs1884302.
Subject(s)
Bone Morphogenetic Protein 2/genetics , Congenital Abnormalities/genetics , Craniosynostoses/genetics , Genetic Predisposition to Disease , Alleles , Animals , Animals, Genetically Modified/genetics , Congenital Abnormalities/physiopathology , Conserved Sequence , Gene Expression Regulation/genetics , Genome-Wide Association Study , Humans , Polymorphism, Single Nucleotide , Regulatory Sequences, Nucleic Acid/genetics , Zebrafish/geneticsABSTRACT
Craniosynostosis (CS) is a major birth defect resulting from premature fusion of cranial sutures. Nonsyndromic CS occurs more frequently than syndromic CS, with sagittal nonsyndromic craniosynostosis (sNCS) presenting as the most common CS phenotype. Previous genome-wide association and targeted sequencing analyses of sNCS have identified multiple associated loci, with the strongest association on chromosome 20. Herein, we report the first whole-genome sequencing study of sNCS using 63 proband-parent trios. Sequencing data for these trios were analyzed using the transmission disequilibrium test (TDT) and rare variant TDT (rvTDT) to identify high-risk rare gene variants. Sequencing data were also examined for copy number variants (CNVs) and de novo variants. TDT analysis identified a highly significant locus at 20p12.3, localized to the intergenic region between BMP2 and the noncoding RNA gene LINC01428. Three variants (rs6054763, rs6054764, rs932517) were identified as potential causal variants due to their probability of being transcription factor binding sites, deleterious combined annotation dependent depletion scores, and high minor allele enrichment in probands. Morphometric analysis of cranial vault shape in an unaffected cohort validated the effect of these three single nucleotide variants (SNVs) on dolichocephaly. No genome-wide significant rare variants, de novo loci, or CNVs were identified. Future efforts to identify risk variants for sNCS should include sequencing of larger and more diverse population samples and increased omics analyses, such as RNA-seq and ATAC-seq.
Subject(s)
Craniosynostoses , Genome-Wide Association Study , Humans , Alleles , Bone Morphogenetic Protein 2/genetics , Craniosynostoses/genetics , DNA, Intergenic/genetics , Whole Genome Sequencing , RNA, Long NoncodingABSTRACT
Frequent benign outgrowths from bone known as osteochondromas, exhibiting typical endochondral ossification, are reported from single to multiple lesions. Characterised by a high incidence of osteochondromas and skeletal deformities, multiple hereditary exostoses (MHE) is the most common inherited musculoskeletal condition. While factors for severity remain unknown, mutations in exostosin 1 and exostosin 2 genes, encoding glycosyltransferases involved in the biosynthesis of ubiquitously expressed heparan sulphate (HS) chains, are associated with MHE. HS-binding bone morphogenetic proteins (BMPs) are multifunctional proteins involved in the morphogenesis of bone and cartilage. HS and HS proteoglycans are involved in BMP-mediated morphogenesis by regulating their gradient formation and activity. Mutations in exostosin genes disturb HS biosynthesis, subsequently affecting its functional role in the regulation of signalling pathways. As BMPs are the primordial morphogens for bone development, we propose the hypothesis that BMP signalling may be critical in osteochondromas. For this reason, the outcomes of exostosin mutations on HS biosynthesis and interactions within osteochondromas and MHE are reviewed. Since BMPs are HS binding proteins, the interactions of HS with the BMP signalling pathway are discussed. The impact of mouse models in the quest to better understand the cell biology of osteochondromas is discussed. Several challenges and questions still remain and further investigations are needed to explore new approaches for better understanding of the pathogenesis of osteochondromas.
Subject(s)
Bone Morphogenetic Proteins/physiology , Bone Neoplasms/physiopathology , Heparitin Sulfate/physiology , Osteochondroma/physiopathology , Signal Transduction/physiology , Animals , Bone Neoplasms/etiology , Disease Models, Animal , Heparitin Sulfate/genetics , Humans , Mice , Mutation/genetics , N-Acetylglucosaminyltransferases/genetics , Osteochondroma/etiologyABSTRACT
Craniosynostosis (CS) is a major birth defect in which one or more skull sutures fuse prematurely. We previously performed a genome-wide association study (GWAS) for sagittal non-syndromic CS (sNCS), identifying associations downstream from BMP2 on 20p12.3 and intronic to BBS9 on 7p14.3; analyses of imputed variants in DLG1 on 3q29 were also genome-wide significant. We followed this work with a GWAS for metopic non-syndromic NCS (mNCS), discovering a significant association intronic to BMP7 on 20q13.31. In the current study, we sequenced the associated regions on 3q29, 7p14.3, and 20p12.3, including two candidate genes (BMP2 and BMPER) near some of these regions in 83 sNCS child-parent trios, and sequenced regions on 7p14.3 and 20q13.2-q13.32 in 80 mNCS child-parent trios. These child-parent trios were selected from the original GWAS cohorts if the probands carried at least one copy of the top associated GWAS variant (rs1884302 C allele for sNCS; rs6127972 T allele for mNCS). Many of the variants sequenced in these targeted regions are strongly predicted to be within binding sites for transcription factors involved in craniofacial development or bone morphogenesis. Variants enriched in more than one trio and predicted to be damaging to gene function are prioritized for functional studies.
Subject(s)
Craniosynostoses , Genome-Wide Association Study , Alleles , Carrier Proteins/genetics , Craniosynostoses/genetics , HumansABSTRACT
PURPOSE: Nonsyndromic craniosynostosis (NCS), the premature fusion of cranial sutures, results in an abnormal skull shape and is associated with a significant morbidity. Proteomics is a promising tool for disease characterization and biomarker discovery; we aimed to identify biologically relevant differentially expressed proteins for NCS. EXPERIMENTAL DESIGN: Label-based quantitative proteomic profiling using TMT was performed on protein extracted from mesenchymal stem cells, osteoblasts and bone tissue of five open and five fused sutures of sagittal NCS (sNCS) and analyzed using quantitative LC-MS/MS based bottom-up proteomics. Differential protein abundance between open and fused sutures was determined to identify biologically relevant proteins of interest. Proteins were validated in an independent sample set by western blot and immunohistochemistry. RESULTS: We observed 838 differentially expressed proteins between open and fused sutures of sNCS. Decorin, lumican, and asporin were significantly downregulated while COL4A1 and TGFß1|1 were upregulated in fused compared to open sutures. CONCLUSIONS AND CLINICAL RELEVANCE: The majority of significantly differentially expressed proteins between open and fused sutures were observed in the proteomes of osteoblasts suggesting that protein changes contributing to premature sagittal suture fusion occur predominantly at the osteoblast level. Our findings suggest a possible ineffective ECM deposition at the osteoblast cell stage.
Subject(s)
ProteomicsABSTRACT
Craniosynostosis (CS), the premature fusion of one or more cranial sutures, is a relatively common congenital anomaly, occurring in 3-5 per 10,000 live births. Nonsyndromic CS (NCS) accounts for up to 80% of all CS cases, yet the genetic factors contributing to the disorder remain largely unknown. The RUNX2 gene, encoding a transcription factor critical for bone and skull development, is a well known CS candidate gene, as copy number variations of this gene locus have been found in patients with syndromic craniosynostosis. In the present study, we aimed to characterize RUNX2 to better understand its role in the genetic etiology and in the molecular mechanisms underlying midline suture ossification in NCS. We report four nonsynonymous variants, one intronic variant and one 18 bp in-frame deletion in RUNX2 not found in our study control population. Significant difference in allele frequency (AF) for the deletion variant RUNX2 p.Ala84-Ala89del (ClinVar 257,095; dbSNP rs11498192) was observed in our sagittal NCS cohort when compared to the general population (P = 1.28 × 10-6), suggesting a possible role in the etiology of NCS. Dual-luciferase assays showed that three of four tested RUNX2 variants conferred a gain-of-function effect on RUNX2, further suggesting their putative pathogenicity in the tested NCS cases. Downregulation of RUNX2 expression was observed in prematurely ossified midline sutures. Metopic sites showed significant downregulation of promoter 1-specific isoforms compared to sagittal sites. Suture-derived mesenchymal stromal cells showed an increased expression of RUNX2 over matched unfused suture derived cells. This demonstrates that RUNX2, and particularly the distal promoter 1-isoform group, are overexpressed in the osteogenic precursors within the pathological suture sites.
Subject(s)
Core Binding Factor Alpha 1 Subunit , Craniosynostoses , Core Binding Factor Alpha 1 Subunit/genetics , Cranial Sutures , Craniosynostoses/genetics , DNA Copy Number Variations , Gain of Function Mutation , HumansABSTRACT
Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-ß. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-ß1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-ß1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-ß1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-ß1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-ß1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-ß1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-ß1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-ß1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-ß1 and exogenous HS.
Subject(s)
Cartilage, Articular/cytology , Chondrocytes/metabolism , Glycoproteins/metabolism , Glycosaminoglycans/pharmacology , Transforming Growth Factor beta/pharmacology , Animals , Cattle , Cell Proliferation/drug effects , Chondrocytes/drug effects , Regeneration/drug effects , Sus scrofaABSTRACT
The expression of bone morphogenetic proteins (BMPs) and their cognate receptors (BMPRs) in osteochondromas has not been investigated. We determined the immunohistochemical localization and distribution of BMP-2/4, -6 and -7; BMP receptors BMPR-1A, BMPR-1B and BMPR-2; signal transducing proteins phosphorylated Smad1/5/8; and BMP antagonist noggin in the cartilaginous cap of solitary (SO) and multiple (MO) human osteochondromas and compared these with bovine growth plate and articular cartilage. The distribution and localization patterns for BMP-6, BMP-7, BMPR-1A and BMPR-2 were similar between the cartilaginous cap and the growth plate. BMP-2/4 and BMPR-1B were present throughout the growth plate. However, BMP-2/4 and phosphorylated Smad1/5/8 were mainly detected in proliferating chondrocytes of the cartilaginous cap. Also, BMPR-1B was found in hypertrophic chondrocytes of SO and proliferating chondrocytes of MO. Noggin was observed in resting chondrocytes and, to a lesser extent, in clustered proliferating chondrocytes in SO. On the other hand, noggin in MO was observed in proliferating chondrocytes. Since BMPs can stimulate proliferation and hypertrophic differentiation of chondrocytes, these findings suggest that there is an imbalance of BMP-2/4 and noggin interactions that may lead to abnormal regulation of chondrocyte proliferation and differentiation in the cartilaginous cap of human osteochondromas.