Identification and Functional Analyses of Molecular Haplotypes of the Human Osteoprotegerin Gene Promoter

Hagedorn, Claudia; Telgmann, Ralph; Dördelmann, Corinna; Schmitz, Boris; Hasenkamp, Sandra; Cambien, François; Paul, Martin; Brand, Eva; Brand-Herrmann, Stefan-Martin

doi:10.1161/atvbaha.109.193532

Cited by 8 publications

(6 citation statements)

References 34 publications

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“…Our observations were supported by a higher frequency of the TT genotype in postmenopausal women with low BMD suggesting that C950T affects bone mass by decreasing OPG expression by osteoblasts/stromal cells in the bone microenvironment. These results agree with those reported by Hagedorn et al (2009), where it was shown that transcriptional factor early growth response (Egr)-1 binds exclusively to the T allele, preventing specific protein (Sp)-1 binding and thus acting as a repressor. However, SNP C950T alleles were not always correlated with serum OPG levels and bone mass, since systemic OPG could be independently influenced by a number of other factors (Ueland et al 2007).…”

Section: Discussionsupporting

confidence: 92%

Functional polymorphisms within the TNFRSF11B (osteoprotegerin) gene increase the risk for low bone mineral density

Vidal

Formosa²,

Xuereb-Anastasi³

2011

Journal of Molecular Endocrinology

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Polymorphisms within the TNFRSF11B gene have been studied and associated with osteoporosis and fracture risk. Osteoprotegerin (OPG), the product of this gene, is a key negative regulator of osteoclastogenesis and is secreted by osteoblasts/stromal cells. A previous study in Maltese postmenopausal women showed positive association of low bone mineral density (BMD) with a polymorphism found within the promoter region of this gene (C950T). In this study, direct DNA sequencing revealed 12 variants with polymorphisms C950T, G1181C and rs4876869 observed to be in strong linkage disequilibrium. The constructed 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; thus, we investigated the functional role of both C950T and rs4876869 in vitro. The promoter region, including the C950T alleles, was amplified by PCR, cloned into pGL3 enhancer vector and transfected into HeLa, COS-7 and RAW264.7 cell lines. After incubation, luciferase activity was measured. The T/C (rs4876869) change was tested for its possible effect on pre-mRNA splicing, using an exon-trapping vector. A statistical significant difference in gene expression was observed between the alleles for T950C, with the T allele showing a lower luciferase expression in all cell lines (P!0 . 01). For rs4876869, exon skipping was observed for the C allele, while only one transcript harbouring the whole exon was observed for the T allele. Our findings suggest that the T-G-T haplotype might be increasing the risk for osteoporosis due to lower quantities of the full OPG transcript being expressed resulting in a higher bone resorption.

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Section: Discussionsupporting

confidence: 92%

Functional polymorphisms within the TNFRSF11B (osteoprotegerin) gene increase the risk for low bone mineral density

Vidal

Formosa²,

Xuereb-Anastasi³

2011

Journal of Molecular Endocrinology

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“…Indeed, it has been very recently reported that the OPG -163 T/C may not affect the expression of the OPG gene 6 , however this was an isolated in vitro report. In this context, there are more SNPs located on the OPG gene 13 , However the data on their functionality and mutual relationships are mostly limited to non-Caucasian populations 21,22 .…”

Section: Discussionmentioning

confidence: 99%

“…It spans 29 kb and consists of 5 exons, 4 introns, and is transcribed into four transcripts, 2.4 kb one representing the major transcript 12 . There is a degree of genetic variation in the gene with at least 10 SNP localised mostly within the 5´flanking region and also exons/introns 13 . A plausible contribution of a genetic variation in the OPG gene to PJI has so far been investigated only in a single centre and with inconsistent results 9,14 .…”

Section: Introductionmentioning

confidence: 99%

Osteoprotegerin gene polymorphism is not associated with prosthetic joint infection after total joint arthroplasty in the Czech population

Navratilova¹,

Gallo²,

Smizansky³

et al. 2014

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub

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a Background and aims. Osteoprotegerin (OPG; official gene symbol: TNFRSF11B) is considered a negative regulator of bone resorption via inhibition of osteoclast differentiation. Further, OPG expression has been detected in Prosthetic Joint Infection (PJI) a serious complication limiting the overall outcome of total joint arthroplasty (TJA). As OPG may be a candidate molecule for PJI pathogenesis, we investigated whether genetic variation in the OPG promoter, namely the SNP at position -163 was associated with PJI. Methods. OPG -163 T/C SNP (rs3102735) was genotyped by polymerase chain reaction with sequence specific primers (PCR-SSP) in 98 Czech patients with PJI and two Czech control groups: 1) aseptic TJA control [251 patients with TJA who did not develop PJI at least 6 yrs. after the surgery] and 2) population control (185 healthy control subjects without TJA). Results. The distribution of OPG -163 SNP genotypes complied with the Hardy-Weinberg equilibrium in all three groups. The allele frequencies of OPG -163 SNP were similar in patients with PJI (minor allele frequency: 0.14), those with aseptic TJA (0.13) and population controls (0.14, P>0.05). Further, there was no significant difference in genotype or phenotype frequency (carriage rate) between patients with PJI and both control groups (P>0.05). Conclusions. In a Czech population, the OPG -163 T/C SNP has not been found to be associated with PJI.

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“…Probe sequences corresponding to the −462 G>A variant in the PPP2R2D promoter region were 5′-biotin-GAATATTACATTGTACAGAA G CCAACAAAAGTTATTGCATAG-3′ for −462 G, and 5′-biotin-GAATATTACATTGTACAGAA A CCAACAAAAGTTATTGCATAG-3′ for −462 A. The consensus sequence of the 5′-biotin-labeled double-stranded probe designed to recognize transcription factor NF1 was 5′-GGGCTAGATGGCTGCCAGCCAAGGCTTCAT-3′ [21]. All labeled and unlabeled oligonucleotides were ordered from Sangon Biotech Co., Ltd (Shanghai, China).…”

Section: Methodsmentioning

confidence: 99%

Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene

et al. 2012

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Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene (PPP2R2D) remain largely unknown. To explore genetic variations in the 5′-flanking region of PPP2R2D gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5′-flanking region of PPP2R2D. Four genetic variants were identified in the 1836 bp 5′-flanking region of PPP2R2D. Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human PPP2R2D promoter luciferase constructs, we found that a 601 bp (−540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5′-flanking fragment revealed the existence of three haplotypes in the distal promoter of PPP2R2D. The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the −462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1). In vitro reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of PPP2R2D proximal promoter. Introduction of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of PPP2R2D gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5′-flanking region of PPP2R2D are critical for transcriptional regulation of PP2A-Bδ.

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Identification and Functional Analyses of Molecular Haplotypes of the Human Osteoprotegerin Gene Promoter

Cited by 8 publications

References 34 publications

Functional polymorphisms within the TNFRSF11B (osteoprotegerin) gene increase the risk for low bone mineral density

Functional polymorphisms within the TNFRSF11B (osteoprotegerin) gene increase the risk for low bone mineral density

Osteoprotegerin gene polymorphism is not associated with prosthetic joint infection after total joint arthroplasty in the Czech population

Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene

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