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Association of CDX1 binding site of periostin gene with bone mineral density and vertebral fracture risk.

Xiao SM, Gao Y, Cheung CL, Bow CH, Lau KS, Sham PC, Tan KC, Kung AW - Osteoporos Int (2012)

Bottom Line: BMD was measured by dual X-ray absorptiometry.The putative transcription factor binding with target sequence was confirmed by electrophoretic mobility shift assay (EMSA).Carriers of the minor allele G per copy of rs9547970 had 1.33 higher risk of vertebral fracture (P = 0.007).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, China. xiaosm@hku.hk

ABSTRACT

Summary: Periostin (POSTN) as a regulator of osteoblast differentiation and bone formation may affect susceptibility to osteoporosis. This study suggests POSTN as a candidate gene for bone mineral density (BMD) variation and vertebral fracture risk, which could better our understanding about the genetic pathogenesis of osteoporosis and will be useful in clinic in the future.

Introduction: The genetic determination of osteoporosis is complex and ill-defined. Periostin (POSTN), an extracellular matrix secreted by osteoblasts and a regulator of osteoblast differentiation and bone formation, may affect susceptibility to osteoporosis.

Methods: We adopted a tag-single nucleotide polymorphism (SNP) based association method followed by imputation-based verification and identification of a causal variant. The association was investigated in 1,572 subjects with extreme-BMD and replicated in an independent population of 2,509 subjects. BMD was measured by dual X-ray absorptiometry. Vertebral fractures were identified by assessing vertebral height from X-rays of the thoracolumbar spine. Association analyses were performed with PLINK toolset and imputation analyses with MACH software. The top imputation finding was subsequently validated by genotyping. Interactions between POSTN and another BMD-related candidate gene sclerostin (SOST) were analyzed using MDR program and validated by logistical regression analyses. The putative transcription factor binding with target sequence was confirmed by electrophoretic mobility shift assay (EMSA).

Results: Several SNPs of POSTN were associated with BMD or vertebral fractures. The most significant polymorphism was rs9547970, located at the -2,327 bp upstream (P = 6.8 × 10(-4)) of POSTN. Carriers of the minor allele G per copy of rs9547970 had 1.33 higher risk of vertebral fracture (P = 0.007). An interactive effect between POSTN and SOST upon BMD variation was suggested (P < 0.01). A specific binding of CDX1 to the sequence of POSTN with the major allele A of rs9547970 but not the variant G allele was confirmed by EMSA.

Conclusions: Our results suggest POSTN as a candidate gene for BMD variation and vertebral fracture risk.

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Electrophoretic mobility shift and competition assays with nuclear extract of HEK293 cells transfected with pCMV-CDX1 and allelic variants of SNP rs9547970 in POSTN. 1 Labeled G probe + nuclear extract(Cdx1−); 2 labeled A probe + nuclear extract (Cdx1−); 3 labeled G probe; 4 labeled G probe + nuclear extract (Cdx1+); 5 labeled G probe + nuclear extract (Cdx1+) + unlabeled G probe; 6 labeled A probe; 7 labeled A probe + nuclear extract (Cdx1+); 8 labeled A probe + nuclear extract (Cdx1+) + unlabeled A probe. Cdx1−, from untreated HEK293; Cdx1+, from HEK293 transfected with pCMV-CDX1
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Fig2: Electrophoretic mobility shift and competition assays with nuclear extract of HEK293 cells transfected with pCMV-CDX1 and allelic variants of SNP rs9547970 in POSTN. 1 Labeled G probe + nuclear extract(Cdx1−); 2 labeled A probe + nuclear extract (Cdx1−); 3 labeled G probe; 4 labeled G probe + nuclear extract (Cdx1+); 5 labeled G probe + nuclear extract (Cdx1+) + unlabeled G probe; 6 labeled A probe; 7 labeled A probe + nuclear extract (Cdx1+); 8 labeled A probe + nuclear extract (Cdx1+) + unlabeled A probe. Cdx1−, from untreated HEK293; Cdx1+, from HEK293 transfected with pCMV-CDX1

Mentions: In the gel shift assay (Fig. 2), the 33-bp oligonucleotides that contained both allelic variants of rs9547970, representing native and mutated CDX1 binding sites, were assayed with nuclear extract of HEK293 cells transfected with pCMV-CDX1. We found a specific binding of CDX1 from nuclear extract of HEK293 cells transfected with pCMV6-CDX1 to the wild-type site centering the rs9547970 major allele A of POSTN. No binding was observed with oligonucleotide containing the minor allele G. Binding to the major A allele resulted in a complex that was specifically competed by 660-fold excess of unlabeled probe containing the major A allele. The results indicate that the A/G change at rs9547970 demolishes a CDX1 binding site in the POSTN gene.Fig. 2


Association of CDX1 binding site of periostin gene with bone mineral density and vertebral fracture risk.

Xiao SM, Gao Y, Cheung CL, Bow CH, Lau KS, Sham PC, Tan KC, Kung AW - Osteoporos Int (2012)

Electrophoretic mobility shift and competition assays with nuclear extract of HEK293 cells transfected with pCMV-CDX1 and allelic variants of SNP rs9547970 in POSTN. 1 Labeled G probe + nuclear extract(Cdx1−); 2 labeled A probe + nuclear extract (Cdx1−); 3 labeled G probe; 4 labeled G probe + nuclear extract (Cdx1+); 5 labeled G probe + nuclear extract (Cdx1+) + unlabeled G probe; 6 labeled A probe; 7 labeled A probe + nuclear extract (Cdx1+); 8 labeled A probe + nuclear extract (Cdx1+) + unlabeled A probe. Cdx1−, from untreated HEK293; Cdx1+, from HEK293 transfected with pCMV-CDX1
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368110&req=5

Fig2: Electrophoretic mobility shift and competition assays with nuclear extract of HEK293 cells transfected with pCMV-CDX1 and allelic variants of SNP rs9547970 in POSTN. 1 Labeled G probe + nuclear extract(Cdx1−); 2 labeled A probe + nuclear extract (Cdx1−); 3 labeled G probe; 4 labeled G probe + nuclear extract (Cdx1+); 5 labeled G probe + nuclear extract (Cdx1+) + unlabeled G probe; 6 labeled A probe; 7 labeled A probe + nuclear extract (Cdx1+); 8 labeled A probe + nuclear extract (Cdx1+) + unlabeled A probe. Cdx1−, from untreated HEK293; Cdx1+, from HEK293 transfected with pCMV-CDX1
Mentions: In the gel shift assay (Fig. 2), the 33-bp oligonucleotides that contained both allelic variants of rs9547970, representing native and mutated CDX1 binding sites, were assayed with nuclear extract of HEK293 cells transfected with pCMV-CDX1. We found a specific binding of CDX1 from nuclear extract of HEK293 cells transfected with pCMV6-CDX1 to the wild-type site centering the rs9547970 major allele A of POSTN. No binding was observed with oligonucleotide containing the minor allele G. Binding to the major A allele resulted in a complex that was specifically competed by 660-fold excess of unlabeled probe containing the major A allele. The results indicate that the A/G change at rs9547970 demolishes a CDX1 binding site in the POSTN gene.Fig. 2

Bottom Line: BMD was measured by dual X-ray absorptiometry.The putative transcription factor binding with target sequence was confirmed by electrophoretic mobility shift assay (EMSA).Carriers of the minor allele G per copy of rs9547970 had 1.33 higher risk of vertebral fracture (P = 0.007).

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong, China. xiaosm@hku.hk

ABSTRACT

Summary: Periostin (POSTN) as a regulator of osteoblast differentiation and bone formation may affect susceptibility to osteoporosis. This study suggests POSTN as a candidate gene for bone mineral density (BMD) variation and vertebral fracture risk, which could better our understanding about the genetic pathogenesis of osteoporosis and will be useful in clinic in the future.

Introduction: The genetic determination of osteoporosis is complex and ill-defined. Periostin (POSTN), an extracellular matrix secreted by osteoblasts and a regulator of osteoblast differentiation and bone formation, may affect susceptibility to osteoporosis.

Methods: We adopted a tag-single nucleotide polymorphism (SNP) based association method followed by imputation-based verification and identification of a causal variant. The association was investigated in 1,572 subjects with extreme-BMD and replicated in an independent population of 2,509 subjects. BMD was measured by dual X-ray absorptiometry. Vertebral fractures were identified by assessing vertebral height from X-rays of the thoracolumbar spine. Association analyses were performed with PLINK toolset and imputation analyses with MACH software. The top imputation finding was subsequently validated by genotyping. Interactions between POSTN and another BMD-related candidate gene sclerostin (SOST) were analyzed using MDR program and validated by logistical regression analyses. The putative transcription factor binding with target sequence was confirmed by electrophoretic mobility shift assay (EMSA).

Results: Several SNPs of POSTN were associated with BMD or vertebral fractures. The most significant polymorphism was rs9547970, located at the -2,327 bp upstream (P = 6.8 × 10(-4)) of POSTN. Carriers of the minor allele G per copy of rs9547970 had 1.33 higher risk of vertebral fracture (P = 0.007). An interactive effect between POSTN and SOST upon BMD variation was suggested (P < 0.01). A specific binding of CDX1 to the sequence of POSTN with the major allele A of rs9547970 but not the variant G allele was confirmed by EMSA.

Conclusions: Our results suggest POSTN as a candidate gene for BMD variation and vertebral fracture risk.

Show MeSH
Related in: MedlinePlus