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Mutations in the EXT1 and EXT2 genes in patients with multiple osteochondromas from Spain
Abstracts / Bone 48 (2011) S153–S160 genes. All data was analysed using the R software. The association between paired miRNA and mRNA profiles was tested by Pearson correlation coefficients and p-values were also computed. Result: A total of 13 miRNAs (miR125b, miR22, miR29b, miR30c, miR31, miR99a, miR140-3p, miR191, miR198, miR335, miR503, miR923 and miR8863p) showed differential expression in primary human bone cells. Statistical analysis of miRNA and mRNA identified a number of genes which were significantly correlated, e.g. PHOSPHO1, FRZB, BMP2, TGFB1 and COL2A. Conclusion: Our results demonstrate that expression of miRNAs exhibiting interindividual variation correlate with mRNAs having known or putative functions in bone metabolism. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
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of them novel), 3 missense (one novel), and one splicing mutation. The four EXT2 mutant alleles were all novel, corresponding to 2 nonsense and 2 frame-shift (small deletions) mutations. All the mutant alleles were different except for the deletion of exons 2–11 in EXT1. We found two of the mutations reported as relatively recurrent: p.Arg340Leu and p. Leu490ArgfsX9. As previously described in other cohorts, most of the mutations (87%) are undoubtedly null. In summary, we have found the molecular bases of all cases but one. Whether this case corresponds to the EXT3 locus remains to be studied. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.343
doi:10.1016/j.bone.2011.03.341
PP183-T No association between variants of the TNFRSF11B gene and Paget's disease of bone N. Alonso⁎, O.M. Albagha, S.H. Ralston Rheumatology Unit, University of Edinburgh, Edinburgh, UK Abstract: Paget's disease of bone (PDB) is a common disorder of the skeleton that affects up to 2% of individuals of European ancestry age 55 years and above. The disease is characterised by focal areas of increased and disorganised bone remodelling that leads to bone pain, bone deformity, pathological fracture, deafness and secondary osteoarthritis. Mutations of SQSTM1 have been identified in about 10% of individuals with “sporadic” PDB and in about 40% of individuals with familial PDB. Juvenile Paget's disease (JPD) is a rare disorder caused by inactivating mutations in the TNFRSF11B gene encoding osteoprotegerin. Mutations in this gene have never been found in patients with typical PDB, but previous studies have reported evidence of an association between common genetic variants of TNFRSF11B and PDB risk in subjects of Belgian and British descent, which was primarily driven by a gender-specific effect in females. Although the TNFRSF11B locus did not emerge as a significant determinant of PDB risk in our recent genome wide association study (Albagha et al., Nat Genet 2010), a genderspecific analysis was not performed. Here we looked for evidence of a gender-specific association between variants in the TNFRSF11B gene and PDB using data from this genome wide association study. We analysed 32 SNPs (12 genotyped and 20 imputed) in 750 PDB patients without SQSTM1 mutations and 2700 controls. The analysed SNPs included all those previously reported in the Belgian and the British cohorts. We performed a gender-specific analysis using PLINK software. Results showed an association between an intronic SNP (rs3134053) and PDB risk in men, but the association was not statistically significant after adjusting for multiple testing. There was no significant association between any of the analysed variants and PDB risk in women and no overall evidence of an association between variants at this locus and PDB. Our data suggest that common variants at the TNFRSF11B locus do not play a major role in regulating susceptibility to PDB. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.342
PP184-S Mutations in the EXT1 and EXT2 genes in patients with multiple osteochondromas from Spain P. Sarrion a,⁎, A. Sangorrin b, R. Urreizti a, R. Artuch b, L. Martorell b, J. Armstrong b, J. Anton b, F. Torner b, M.A. Vilaseca b, S. Balcells a, D. Grinberg a a Dept Genetics, University of Barcelona, CIBERER, IBUB, Barcelona, Spain b Hospital Sant Joan de Déu, Barcelona, Spain Abstract: Multiple osteochondromas (MO) is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours (exostoses osteochondromas). Osteochondromas are the result of excessive chondrocyte proliferation and bone growth at the juxtaepiphyseal regions of long bones. Osteochondromas are the most common benign bone tumour. Although benign, they can cause short stature, several secondary complications and in some cases malignant transformation. Three loci have been identified to date, EXT1, mapped to 8q24, EXT2 on 11p11–p13, and EXT3 on 19p, but most of the cases are caused by mutations in either the EXT1 (around 65%) or EXT2 (more than 30%) genes, while the EXT3 gene remains to be identified. In this study we present a mutation analysis of the EXT1 and EXT2 genes in 18 patients, most of them with moderate phenotype. One patient presented with malignant transformation to chondrosarcoma. The mutation analysis was carried out sequencing exons and flanking regions of EXT1 and EXT2, and performing MLPA analysis. We have found 17 mutant alleles (94%), 13 in EXT1 and 4 in EXT2, 8 of them novel. Four of the EXT1 mutations were deletions that were identified by MLPA: one involved the deletion of the whole gene, two deletions spanned from exon 2 to exon 11, while the fourth case involved the deletion of exon 8. The remaining 9 EXT1 mutations were the following point mutations: 2 nonsense (both novel), 3 frame-shift (small deletions, one
PP185-M Genetic variants in the optineurin gene are associated with disease severity in Paget's disease of bone R. Obaid a,⁎, W.D. Fraser b, P.L. Selby c, S.H. Ralston a, O.M. Albagha a a Molecular Medicine Centre, University of Edinburgh, Edinburgh, UK b Department of Clinical Chemistry, Royal Liverpool University Hospital, Liverpool, UK c Department of Medicine, University of Manchester, Manchester, UK Abstract: Paget's disease of bone (PDB) is a common disease characterised by focal increases in bone remodelling leading to bone pain and other clinical complications such as bone deformity, fracture, deafness, and osteoarthritis. Mutations in SQSTM1 gene are known to cause about 10% of PDB cases and previous studies have shown that SQSTM1 mutations are associated with disease severity (Visconti et al. JBMR 2010). Recent genome wide association studies identified further susceptibility genes for PDB including CSF1, OPTN, and TNFRSF11A (Albagha et al. Nat. Genet. 2010). The risk allele “T” of the common variant rs1561570 in the OPTN gene was associated with PDB risk (P = 6.9 × 10 −13; OR = 1.5). OPTN encodes optineurin, a ubiquitous cytoplasmic protein with possible role in the NFkB signalling pathway, but its role in bone metabolism is yet to be defined. In this study we investigated the relationship between rs1561570 in the OPTN gene and disease severity in 635 PDB patients without SQSTM1 mutations derived from the PRISM trial. A disease severity score was devised based on several clinical features including: number of affected bones, clinical evidence of bone deformity, the presence of bone pain, bone fractures, requirement of orthopaedic surgery, and the use of hearing aid for deafness. The association between rs1561570 and disease severity score was investigated using general linear model ANOVA adjusting for age and gender. Analysis results showed significant association between rs1561570 and severity score so that carriers of the risk allele “T” had higher severity score (6.02 ± 0.11) compared to non-carriers (5.39 ± 0.11; P = 0.031). We also found a trend for reduced quality of life (as assessed by SF36 physical summary score) in patients who carried the risk allele (36.8 ± 1.41) compared to those who did not (39.3 ± 0.5; P = 0.09). In conclusion, our results showed that OPTN gene variants are associated with disease severity and complications of PDB. These findings could be of clinical value in identifying patients (who are SQSTM1 negative) at risk of developing severe disease, however further studies in larger cohorts will be required to confirm these findings. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.344
PP186-T Functional polymorphisms in the TNFRSF11B (osteoprotegerin) gene and low bone mineral density in postmenopausal women in Malta R. Formosa⁎, C. Vidal, A. Xuereb-Anastasi Faculty of Health Science, Department of Applied Biomedical Science, University of Malta, Msida, Malta Abstract: Polymorphisms within the TNFRSF11B gene have been studied and associated with osteoporosis and fracture risk. In a previous study, we sequenced the coding and promoter regions of this gene in a group of postmenopausal women in Malta with normal and low bone mineral density (BMD). Polymorphisms T950C, G1181C and rs4876869 were in strong linkage disequilibrium and haplotype T–G–T was found to increase the risk for a low BMD, while C–G–T and C–C–C have a protective role. In this study, we investigated the functional role of both T950C and rs4876869 in vitro. The T950C in the promoter region was investigated for its effect on gene expression using a luciferase reporter assay technique. The promoter region, including the T950C alleles, was amplified by PCR and cloned into pGL3 enhancer vector. Constructs were transfected into HeLa and RAW264.7 cell lines and incubated for 48 and 24 hours, respectively, at 37 °C in 5% CO2. After incubation, cells were lysed, and luciferase activity was measured. The T/C (rs4876869) change found in intron 2 was tested for its possible effect on pre-mRNA splicing using the exon-trapping vector pSPL3. The whole region harbouring the polymorphism was
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of them novel), 3 missense (one novel), and one splicing mutation. The four EXT2 mutant alleles were all novel, corresponding to 2 nonsense and 2 frame-shift (small deletions) mutations. All the mutant alleles were different except for the deletion of exons 2–11 in EXT1. We found two of the mutations reported as relatively recurrent: p.Arg340Leu and p. Leu490ArgfsX9. As previously described in other cohorts, most of the mutations (87%) are undoubtedly null. In summary, we have found the molecular bases of all cases but one. Whether this case corresponds to the EXT3 locus remains to be studied. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared. doi:10.1016/j.bone.2011.03.343
doi:10.1016/j.bone.2011.03.341
PP183-T No association between variants of the TNFRSF11B gene and Paget's disease of bone N. Alonso⁎, O.M. Albagha, S.H. Ralston Rheumatology Unit, University of Edinburgh, Edinburgh, UK Abstract: Paget's disease of bone (PDB) is a common disorder of the skeleton that affects up to 2% of individuals of European ancestry age 55 years and above. The disease is characterised by focal areas of increased and disorganised bone remodelling that leads to bone pain, bone deformity, pathological fracture, deafness and secondary osteoarthritis. Mutations of SQSTM1 have been identified in about 10% of individuals with “sporadic” PDB and in about 40% of individuals with familial PDB. Juvenile Paget's disease (JPD) is a rare disorder caused by inactivating mutations in the TNFRSF11B gene encoding osteoprotegerin. Mutations in this gene have never been found in patients with typical PDB, but previous studies have reported evidence of an association between common genetic variants of TNFRSF11B and PDB risk in subjects of Belgian and British descent, which was primarily driven by a gender-specific effect in females. Although the TNFRSF11B locus did not emerge as a significant determinant of PDB risk in our recent genome wide association study (Albagha et al., Nat Genet 2010), a genderspecific analysis was not performed. Here we looked for evidence of a gender-specific association between variants in the TNFRSF11B gene and PDB using data from this genome wide association study. We analysed 32 SNPs (12 genotyped and 20 imputed) in 750 PDB patients without SQSTM1 mutations and 2700 controls. The analysed SNPs included all those previously reported in the Belgian and the British cohorts. We performed a gender-specific analysis using PLINK software. Results showed an association between an intronic SNP (rs3134053) and PDB risk in men, but the association was not statistically significant after adjusting for multiple testing. There was no significant association between any of the analysed variants and PDB risk in women and no overall evidence of an association between variants at this locus and PDB. Our data suggest that common variants at the TNFRSF11B locus do not play a major role in regulating susceptibility to PDB. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.342
PP184-S Mutations in the EXT1 and EXT2 genes in patients with multiple osteochondromas from Spain P. Sarrion a,⁎, A. Sangorrin b, R. Urreizti a, R. Artuch b, L. Martorell b, J. Armstrong b, J. Anton b, F. Torner b, M.A. Vilaseca b, S. Balcells a, D. Grinberg a a Dept Genetics, University of Barcelona, CIBERER, IBUB, Barcelona, Spain b Hospital Sant Joan de Déu, Barcelona, Spain Abstract: Multiple osteochondromas (MO) is an autosomal dominant skeletal disorder characterized by the formation of multiple cartilage-capped tumours (exostoses osteochondromas). Osteochondromas are the result of excessive chondrocyte proliferation and bone growth at the juxtaepiphyseal regions of long bones. Osteochondromas are the most common benign bone tumour. Although benign, they can cause short stature, several secondary complications and in some cases malignant transformation. Three loci have been identified to date, EXT1, mapped to 8q24, EXT2 on 11p11–p13, and EXT3 on 19p, but most of the cases are caused by mutations in either the EXT1 (around 65%) or EXT2 (more than 30%) genes, while the EXT3 gene remains to be identified. In this study we present a mutation analysis of the EXT1 and EXT2 genes in 18 patients, most of them with moderate phenotype. One patient presented with malignant transformation to chondrosarcoma. The mutation analysis was carried out sequencing exons and flanking regions of EXT1 and EXT2, and performing MLPA analysis. We have found 17 mutant alleles (94%), 13 in EXT1 and 4 in EXT2, 8 of them novel. Four of the EXT1 mutations were deletions that were identified by MLPA: one involved the deletion of the whole gene, two deletions spanned from exon 2 to exon 11, while the fourth case involved the deletion of exon 8. The remaining 9 EXT1 mutations were the following point mutations: 2 nonsense (both novel), 3 frame-shift (small deletions, one
PP185-M Genetic variants in the optineurin gene are associated with disease severity in Paget's disease of bone R. Obaid a,⁎, W.D. Fraser b, P.L. Selby c, S.H. Ralston a, O.M. Albagha a a Molecular Medicine Centre, University of Edinburgh, Edinburgh, UK b Department of Clinical Chemistry, Royal Liverpool University Hospital, Liverpool, UK c Department of Medicine, University of Manchester, Manchester, UK Abstract: Paget's disease of bone (PDB) is a common disease characterised by focal increases in bone remodelling leading to bone pain and other clinical complications such as bone deformity, fracture, deafness, and osteoarthritis. Mutations in SQSTM1 gene are known to cause about 10% of PDB cases and previous studies have shown that SQSTM1 mutations are associated with disease severity (Visconti et al. JBMR 2010). Recent genome wide association studies identified further susceptibility genes for PDB including CSF1, OPTN, and TNFRSF11A (Albagha et al. Nat. Genet. 2010). The risk allele “T” of the common variant rs1561570 in the OPTN gene was associated with PDB risk (P = 6.9 × 10 −13; OR = 1.5). OPTN encodes optineurin, a ubiquitous cytoplasmic protein with possible role in the NFkB signalling pathway, but its role in bone metabolism is yet to be defined. In this study we investigated the relationship between rs1561570 in the OPTN gene and disease severity in 635 PDB patients without SQSTM1 mutations derived from the PRISM trial. A disease severity score was devised based on several clinical features including: number of affected bones, clinical evidence of bone deformity, the presence of bone pain, bone fractures, requirement of orthopaedic surgery, and the use of hearing aid for deafness. The association between rs1561570 and disease severity score was investigated using general linear model ANOVA adjusting for age and gender. Analysis results showed significant association between rs1561570 and severity score so that carriers of the risk allele “T” had higher severity score (6.02 ± 0.11) compared to non-carriers (5.39 ± 0.11; P = 0.031). We also found a trend for reduced quality of life (as assessed by SF36 physical summary score) in patients who carried the risk allele (36.8 ± 1.41) compared to those who did not (39.3 ± 0.5; P = 0.09). In conclusion, our results showed that OPTN gene variants are associated with disease severity and complications of PDB. These findings could be of clinical value in identifying patients (who are SQSTM1 negative) at risk of developing severe disease, however further studies in larger cohorts will be required to confirm these findings. This article is part of a Special Issue entitled ECTS 2011. Disclosure of interest: None declared.
doi:10.1016/j.bone.2011.03.344
PP186-T Functional polymorphisms in the TNFRSF11B (osteoprotegerin) gene and low bone mineral density in postmenopausal women in Malta R. Formosa⁎, C. Vidal, A. Xuereb-Anastasi Faculty of Health Science, Department of Applied Biomedical Science, University of Malta, Msida, Malta Abstract: Polymorphisms within the TNFRSF11B gene have been studied and associated with osteoporosis and fracture risk. In a previous study, we sequenced the coding and promoter regions of this gene in a group of postmenopausal women in Malta with normal and low bone mineral density (BMD). Polymorphisms T950C, G1181C and rs4876869 were in strong linkage disequilibrium and haplotype T–G–T was found to increase the risk for a low BMD, while C–G–T and C–C–C have a protective role. In this study, we investigated the functional role of both T950C and rs4876869 in vitro. The T950C in the promoter region was investigated for its effect on gene expression using a luciferase reporter assay technique. The promoter region, including the T950C alleles, was amplified by PCR and cloned into pGL3 enhancer vector. Constructs were transfected into HeLa and RAW264.7 cell lines and incubated for 48 and 24 hours, respectively, at 37 °C in 5% CO2. After incubation, cells were lysed, and luciferase activity was measured. The T/C (rs4876869) change found in intron 2 was tested for its possible effect on pre-mRNA splicing using the exon-trapping vector pSPL3. The whole region harbouring the polymorphism was